Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair

ABSTRACT

Provided herein include methods and compositions for effecting a targeted genetic change in DNA in a cell. Certain aspects and embodiments relate to improving the efficiency of the targeting of modifications to specific locations in genomic or other nucleotide sequences. As described herein, nucleic acids which direct specific changes to the genome may be combined with various approaches to enhance the availability of components of the natural repair systems present in the cells being targeted for modification.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/966,952, filed Apr. 29, 2018, which issued as U.S. Pat. No.10,954,522, which is a continuation of U.S. patent application Ser. No.15/069,885, filed Mar. 14, 2016, which issued as U.S. Pat. No.9,957,515, on May 1, 2018, which is a continuation-in-part applicationof International Patent Application No. PCT/US2015/020622, filed Mar.14, 2015, which claims priority to U.S. Provisional Application No.61/953,333, filed Mar. 14, 2014; U.S. Provisional Application No.62/051,579, filed Sep. 17, 2014; U.S. Provisional Application No.62/075,811, filed Nov. 5, 2014; U.S. Provisional Application No.62/075,816, filed Nov. 5, 2014; and U.S. Provisional Application No.62/133,129, filed Mar. 13, 2015; and is a continuation-in-partapplication of U.S. patent application Ser. No. 14/777,357, which is aU.S. National Phase Application of International Patent Application No.PCT/US2014/029566, filed Mar. 14, 2014, which claims priority to U.S.Provisional Application No. 61/801,333, filed Mar. 15, 2013, each ofwhich is hereby incorporated by reference in its entirety including alltables, figures and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 22, 2021, isnamed CIBUS-029-CT2_SeqListing.txt and is 220 kilobytes in size.

FIELD OF THE INVENTION

The instant disclosure relates at least in part to targeted geneticmutations and modifications, including methods and compositions formaking such mutations and modifications.

BACKGROUND

The following discussion is merely provided to aid the reader inunderstanding and is not admitted to describe or constitute prior art tothe present disclosure.

U.S. Pat. No. 6,271,360 discloses methods and compositions for theintroduction of predetermined genetic changes in target genes of aliving cell by introducing an oligodeoxynucleotide encoding thepredetermined change. The oligodeoxynucleotides are effective inmammalian, avian, plant and bacterial cells.

U.S. Pat. No. 8,771,945 discloses vectors and vector systems, some ofwhich encode one or more components of a CRISPR complex, as well asmethods for the design and use of such vectors.

U.S. Pat. No. 8,470,973 “refers to methods for selectively recognizing abase pair in a DNA sequence by a polypeptide, to modified polypeptideswhich specifically recognize one or more base pairs in a DNA sequenceand, to DNA which is modified so that it can be specifically recognizedby a polypeptide and to uses of the polypeptide and DNA in specific DNAtargeting as well as to methods of modulating expression of target genesin a cell.”

SUMMARY

Provided herein include methods and compositions for effecting atargeted genetic change in DNA in a cell. Certain aspects andembodiments relate to improving the efficiency of the targeting ofmodifications to specific locations in genomic or other nucleotidesequences. As described herein, nucleic acids which direct specificchanges to the genome may be combined with various approaches to enhancethe availability of components of the natural repair systems present inthe cells being targeted for modification.

In a first aspect, provided are methods for introducing a gene repairoligonucleobase (GRON)-mediated mutation into a target deoxyribonucleicacid (DNA) sequence in a plant cell. In certain embodiments the methodsmay include, inter alia, culturing the plant cell under conditions thatincrease one or more cellular DNA repair processes prior to, and/orcoincident with, delivery of a GRON into the plant cell; and/or deliveryof a GRON into the plant cell greater than 15 bases in length, the GRONoptionally comprising one or more; or two or more; mutation sites forintroduction into the target DNA.

A “gene repair oligonucleotide” or “GRON” as used herein means anoligonucleobase (e.g., mixed duplex oligonucleotides, non-nucleotidecontaining molecules, single stranded oligodeoxynucleotides, doublestranded oligodeoxynucleotides and other gene repair molecules) that canunder certain conditions direct single, or in some embodiments multiple,nucleotide deletions, insertions or substitutions in a DNA sequence.This oligonucleotide-mediated gene repair editing of the genome maycomprise both non-homology based repair systems (e.g., non-homologousend joining) and homology-based repair systems (e.g., homology-directedrepair). The GRON is typically designed to align in register with agenomic target except for the designed mismatch(es). These mismatchescan be recognized and corrected by harnessing one or more of the cell'sendogenous DNA repair systems. In some embodiments a GRON oroligonucleotide can be designed to contain multiple differences whencompared to the organisms target sequence. These differences may not allaffect the protein sequence translated from said target sequence and inone or more cases be known as silent changes. Numerous variations ofGRON structure, chemistry and function are described elsewhere herein.In various embodiments, a GRON as used herein may have one or moremodifications. For example, a GRON as used herein may have one or moremodifications that attract DNA repair machinery to the targeted(mismatch) site and/or that prevent recombination of part or all of theGRON (other than the desired targeted deletion(s), insertion(s),substitution(s) or the like) into the genomic DNA of the target DNAsequence and/or that increase the stability of the GRON.

In various embodiments, a GRON may have both RNA and DNA nucleotidesand/or other types of nucleobases. In some embodiments, one or more ofthe DNA or RNA nucleotides comprise a modification.

In one aspect, provided is a method of causing a genetic change in aplant cell, wherein the method involves exposing the cell to a DNAcutter and a GRON, for example a GRON that is modified as contemplatedherein. In some embodiments the GRON may be modified such as with a Cy3group, 3PS group, a 2′O-methyl group or other modification such ascontemplated herein. In another aspect, provided is a plant cell thatincludes a DNA cutter and a GRON, for example where the GRON is modifiedsuch as with a Cy3 group, 3PS group, a 2′O-methyl group or othermodification. In some embodiments, the DNA cutter is one or moreselected from a CRISPR, a TALEN, a zinc finger, meganuclease, and aDNA-cutting antibiotic. In some embodiments, the DNA cutter is a CRISPR.In some embodiments, the DNA cutter is a TALEN. In some embodiments, theGRON is between 15 and 60 nucleobases in length; or between 30 and 40nucleobases in length; or between 35 and 45 nucleobases in length; orbetween 20 and 70 nucleobases in length; or between 20 and 200nucleobases in length; or between 30 and 180 nucleobases in length; orbetween 50 and 160 nucleobases in length; or between 70 and 150nucleobases in length; or between 70 and 210 nuceleobases in length; orbetween 80 and 120 nucleobases in length; or between 90 and 110nucleobases in length; or between 95 and 105 nucleobases in length; orbetween 80 and 300 nucleobases in length; or between 90 and 250nucleobases in length; or between 100 and 150 nucleobases in length; orbetween 100 and 200 nucleobases in length; or between 100 and 210nucleobases in length; or between 100 and 300 nucleobases in length; orbetween 150 and 200 nucleobases in length; or between 200 and 300nucleobases in length; or between 250 and 350 nucleobases in length; orbetween 50 and 110 nucleobases in length; or between 50 and 200nucleobases in length; or between 150 and 210 nucleobases in length; orbetween 20 and 1000 nucleobases in length; or between 100 and 1000nucleobases in length; or between 200 and 1000 nucleobases in length; orbetween 300 and 1000 nucleobases in length; or between 400 and 1000nucleobases in length; or between 500 and 1000 nucleobases in length; orbetween 600 and 1000 nucleobases in length; or between 700 and 1000nucleobases in length; or between 800 and 1000 nucleobases in length; orbetween 900 and 1000 nucleobases in length; or between 300 and 800nucleobases in length; or between 400 and 600 nucleobases in length; orbetween 500 and 700 nucleobases in length; or between 600 and 800nucleobases in length; or longer than 30 nucleobases in length; orlonger than 35 nucleobases in length; or longer than 40 nucleobases inlength; or longer than 50 nucleobases in length; or longer than 60nucleobases in length; or longer than 65 nucleobases in length; orlonger than 70 nucleobases in length; or longer than 75 nucleobases inlength; or longer than 80 nucleobases in length; or longer than 85nucleobases in length; or longer than 90 nucleobases in length; orlonger than 95 nucleobases in length; or longer than 100 nucleobases inlength; or longer than 110 nucleobases in length; or longer than 125nucleobases in length; or longer than 150 nucleobases in length; orlonger than 165 nucleobases in length; or longer than 175 nucleobases inlength; or longer than 200 nucleobases in length; or longer than 250nucleobases in length; or longer than 300 nucleobases in length; orlonger than 350 nucleobases in length; or longer than 400 nucleobases inlength; or longer than 450 nucleobases in length; or longer than 500nucleobases in length; or longer than 550 nucleobases in length; orlonger than 600 nucleobases in length; or longer than 700 nucleobases inlength; or longer than 800 nucleobases in length; or longer than 900nucleobases in length. GRONs may be targeted at both non-coding (NC) andcoding (C) regions of a target gene. By way of example, FIGS. 27 and 28respectively depict C-GRONs and NC-GRONs suitable for introducingmutations into the rice genome in order to introduce one or more of thefollowing amino acid substitions to the ACCase gene. The convention isto use the amino acid numbering system for the plastidal ACCase fromblackgrass (Alopecurus myosuroides; Am) as the reference. The ACCasenumbering used herein is based on the numbering for the blackgrassreference sequence ACCase protein (SEQ ID NO: 1) or at an analogousamino acid residue in an ACCase paralog (V=CY3; H=3′DMT dC CPG). Thefollowing table lists ACCase mutations that produce one or more ofalloxydim, butroxydim, clethodim, cloproxydim, cycloxydim, sethoxydim,tepraloxydim, tralkoxydim, chlorazifop, clodinafop, clofop, diclofop,fenoxaprop, fenoxaprop-P, fenthiaprop, fluazifop, fluazifop-P,haloxyfop, haloxyfop-P, isoxapyrifop, propaquizafop, quizalofop,quizalofop-P, trifop, pinoxaden, agronomically acceptable salts andesters of any of these herbicides, and combinations thereof resistantphenotype.

Amino Amino Acid Acid Change Codon Change Change Codon Change I1781AATA > GCT C2088F TGC > TTT ATA > GCC TGC > TTC ATA > GCA ATA > GCGC2088G TGC > GGT TGC > GGC I1781L ATA > CTT TGC > GGA ATA > CTC TGC >GGG ATA > CTA ATA > CTG C2088H TGC > CAT ATA > TTA TGC > CAC ATA > TTGC2088K TGC > AAA I1781M ATA > ATG TGC > AAG I1781N ATA > AAT C2088LTGC > CTT ATA > AAC TGC > CTC TGC > CTA I1781S ATA > TCT TGC > CTG ATA >TCC TGC > TTA ATA > TCA TGC > TTG ATA > TCG C2088N TGC > AAT I1781TATA > ACT TGC > AAC ATA > ACC ATA > ACA C2088P TGC > CCT ATA > ACG TGC >CCC TGC > CCA I1781V ATA > GTT TGC > CCG ATA > GTC ATA > GTA C2088QTGC > CAA ATA > GTG TGC > CAG G1783C GGA > TGT C2088R TGC > CGT GGA >TGC TGC > CGC TGC > CGA A1786P GCT > CCT TGC > CGG GCT > CCC TGC > AGAGCT > CCA TGC > AGG GCT > CCG C2088S TGC > TCT D2078G GAT > GGT TGC >TCC GAT > GGC TGC > TCA GAT > GGA TGC > TCG GAT > GGG C2088T TGC > ACTD2078K GAT > AAA TGC > ACC GAT > AAG TGC > ACA TGC > ACG D2078T GAT >ACT GAT > ACC C2088V TGC > GTT GAT > ACA TGC > GTC GAT > ACG TGC > GTATGC > GTG S2079F AGC> TTT AGC > TTC C2088W TGC > TGG K2080E AAG > GAAAAG > GAG

Similarly, FIGS. 29 and 30 respectively depict (coding) C-GRONs and(non-coding) NC-GRONs suitable for introducing mutations into the flaxgenome in order to introduce one or more of the following amino acidsubstitions to the EPSPS gene (with all numbering relative to the aminoacid sequence of the E. coli AroA protein (prokaryotic EPSPS equivalent)(such as those described in U.S. Pat. No. 8,268,622). (V=CY3; H=3′DMT dCCPG). The following table lists EPSPS mutations that produce glyphosateagronomically acceptable salts and esters of any of these herbicides,and combinations thereof resistant phenotype.

Amino Acid Change Codon Change G96A GGA > GCT GGA > GCC GGA > GCA GGA >GCG T97I ACA > ATT ACA > ATC ACA > ATA P101A CCG > GCT CCG > GCC CCG >GCA CCG > GCG P101S CCG > TCT CCG > TCC CCG > TCA CCG > TCG P101T CCG >ACT CCG > ACC CCG > ACA CCG > ACG

The term “CRISPR” as used herein refers to elements; i.e., a cas (CRISPRassociated) gene, transcript (e.g., mRNA) or protein and at least oneCRISPR spacer sequence (Clustered Regularly Interspaced ShortPalindromic Repeats, also known as SPIDRs—SPacer Interspersed DirectRepeats); that when effectively present or expressed in a cell couldeffect cleavage of a target DNA sequence via CRISPR/CAS cellularmachinery such as described in e.g., Cong, L. et al., Science, vol. 339no 6121 pp. 819-823 (2013); Jinek et al, Science, vol. 337:816-821(2013); Wang et al., RNA, vol. 14, pp. 903-913 (2008); Zhang et al.,Plant Physiology, vol. 161, pp. 20-27 (2013), Zhang et al, PCTApplication No. PCT/US2013/074743; and Charpentier et al., PCTApplication No. PCT/US2013/032589. In some embodiments, such as forexample a CRISPR for use in a eukaryotic cell, a CRISPR as contemplatedherein may also include an additional element that includes a sequencefor one or more functional nuclear localization signals. CRISPRs ascontemplated herein can be expressed in, administered to and/or presentin a cell (such as a plant cell) in any of many ways or manifestations.For example a CRISPR as contemplated herein may include or involve oneor more of a CRISPR on a plasmid, a CRISPR nickase on a plasmid, aCRISPRa on a plasmid, or a CRISPRi on a plasmid as follows:

CRISPR on a plasmid: A recombinant expression vector comprising:

-   -   (i) a nucleotide sequence encoding a DNA-targeting RNA (e.g.,        guide RNA), wherein the DNA-targeting RNA comprises:        -   a. a first segment comprising a nucleotide sequence that is            complementary to a sequence in a target DNA (e.g.,            protospacer, spacer, or crRNA); and        -   b. a second segment that interacts with a site-directed            modifying polypeptide (e.g., trans-activating crRNA or            tracrRNA); and    -   (ii) a nucleotide sequence encoding the site-directed modifying        polypeptide (e.g., cas gene), wherein the site-directed        polypeptide comprises:        -   a. an RNA-binding portion that interacts with the            DNA-targeting RNA (e.g., REC lobe); and        -   b. an activity portion that causes double-stranded breaks            within the target DNA (e.g., NUC lobe), wherein the site of            the double-stranded breaks within the target DNA is            determined by the DNA-targeting RNA.

CRISPR nickase on a plasmid. A recombinant expression vector comprising:

-   -   (i) a nucleotide sequence encoding a DNA-targeting RNA (e.g.,        guide RNA), wherein the DNA-targeting RNA comprises:        -   a. a first segment comprising a nucleotide sequence that is            complementary to a sequence in a target DNA (e.g.,            protospacer, spacer, or crRNA); and        -   b. a second segment that interacts with a site-directed            modifying polypeptide (e.g., trans-activating crRNA or            tracrRNA); and    -   (ii) a nucleotide sequence encoding the site-directed modifying        polypeptide (e.g., cas gene), wherein the site-directed        polypeptide comprises:        -   a. an RNA-binding portion that interacts with the            DNA-targeting RNA (e.g., REC lobe); and        -   b. an activity portion that causes single-stranded breaks            within the target DNA (e.g., NUC lobe), wherein the site of            the single-stranded breaks within the target DNA is            determined by the DNA-targeting RNA.

CRISPRa on a plasmid. A recombinant expression vector comprising:

-   -   (i) a nucleotide sequence encoding a DNA-targeting RNA (e.g.,        guide RNA), wherein the DNA-targeting RNA comprises:        -   a. a first segment comprising a nucleotide sequence that is            complementary to a sequence in a target DNA (e.g.,            protospacer, spacer, or crRNA); and        -   b. a second segment that interacts with a site-directed            modifying polypeptide (e.g., trans-activating crRNA or            tracrRNA); and    -   (ii) a nucleotide sequence encoding the site-directed modifying        polypeptide (e.g., cas gene), wherein the site-directed        polypeptide comprises:        -   a. an RNA-binding portion that interacts with the            DNA-targeting RNA (e.g., REC lobe); and        -   b. an activity portion that modulates transcription (e.g.,            NUC lobe; in certain embodiments increases transcription)            within the target DNA, wherein the site of the            transcriptional modulation within the target DNA is            determined by the DNA-targeting RNA.

CRISPRi on a plasmid. A recombinant expression vector comprising:

-   -   (i) a nucleotide sequence encoding a DNA-targeting RNA (e.g.,        guide RNA), wherein the DNA-targeting RNA comprises:        -   a. a first segment comprising a nucleotide sequence that is            complementary to a sequence in a target DNA (e.g.,            protospacer, spacer, or crRNA); and        -   b. a second segment that interacts with a site-directed            modifying polypeptide (e.g., trans-activating crRNA or            tracrRNA); and    -   (ii) a nucleotide sequence encoding the site-directed modifying        polypeptide (e.g., cas gene), wherein the site-directed        polypeptide comprises:        -   a. an RNA-binding portion that interacts with the            DNA-targeting RNA (e.g., REC lobe); and        -   b. an activity portion that modulates            transcription/translation (e.g., NUC lobe; in some            embodiments decreases transcription/translation) within the            target DNA, wherein the site of            transcriptional/translational modulation within the target            DNA is determined by the DNA-targeting RNA.

Each of the CRISPR on a plasmid, CRISPR nickase on a plasmid, CRISPRa ona plasmid, and CRISPRi on a plasmid may in some embodimentsalternatively have one or more appropriate elements be administered,expressed or present in a cell as an RNA (e.g., mRNA) or a proteinrather than on a plasmid. Delivery of protected mRNA may be as describedin Kariko, et al, U.S. Pat. No. 8,278,036.

In some embodiments, each of the CRISPRi and CRISPRa may include adeactivated cas9 (dCas9). A deactivated cas9 still binds to target DNA,but does not have cutting activity. Nuclease-deficient Cas9 can resultfrom D10A and H840A point mutations which inactivates its two catalyticdomains.

In some embodiments, a CRISPRi inhibits transcription initiation orelongation via steric hindrance of RNA Polymerase II. CRISPRi canoptionally be enhanced (CRISPRei) by fusion of a strong repressor domainto the C-terminal end of a dCas9 protein. In some embodiments, arepressor domain recruits and employs chromatin modifiers. In someembodiments, the repressor domain may include, but is not limited todomains as described in Kagale, S. et al., Epigenetics, vol. 6 no 2pp141-146 (2011):

1. (SEQ ID NO: 3) LDLNRPPPVEN (SEQ ID NO: 4)OsERF3 repressor domain (LxLxPP motif) 2. (SEQ ID NO: 5) LRLFGVNM(R/KLFGV motif (SEQ ID NO: 6)) AtBRD repressor domain 3. (SEQ ID NO: 7)LKLFGVWL (R/KLFGV motif (SEQ ID NO: 6)) AtHsfB1 repressor domain 4.(SEQ ID NO: 8) LDLELRLGFA AtSUP repressor domain (EAR motif) 5.(SEQ ID NO: 9) ERSNSIELRNSFYGRARTSPWSYGDYDNCQQDHDYLLGFSWPPRSYTCSFCKREFRSAQALGGHMNVHR RDRARLRLQQSPSSSSTPSPPYPNPNYSYSTMANSPPPHHSPLTLFPTLSPPSSPRYRAGLIRSLSPKSK HTPENACKTKKSSLLVEAGEATRFTSKDACKILRNDEIISLELEIGLINESEQDLDLELRLGFA* full AtSUP gene containing repressordomain (EAR motif)

In some embodiments, a CRISPRa activation of transcription achieved byuse of dCas9 protein containing a fused C-terminal end transcriptionalactivator. In some embodiments, an activation may include, but is notlimited to VP64 (4X VP16), AtERF98 activation domain, or AtERF98×4concatemers such as described in Cheng, AW et al., Cell Research, pp1-9(2013); Perez-Pinera, P. et al., Nature Methods, vol. 10 pp 913-976(2013); Maeder, ML. et al., Nature Methods, vol. 10 pp 977-979 (2013)and Mali, P., et al., Nature Biotech., vol. 31 pp 833-838 (2013).

In some embodiments the CRISPR includes a nickase. In certainembodiments, two or more CRISPR nickases are used. In some embodiments,the two or more nickases cut on opposite strands of target nucleic acid.In other embodiments, the two or more nickases cut on the same strand oftarget nucleic acid.

As used herein, “repressor protein” or “repressor” refers to a proteinthat binds to operator of DNA or to RNA to prevent transcription ortranslation, respectively.

As used herein, “repression” refers to inhibition of transcription ortranslation by binding of repressor protein to specific site on DNA ormRNA. In some embodiments, repression includes a significant change intranscription or translation level of at least 1.5 fold, in otherembodiments at least two fold, and in other embodiments at least fivefold.

As used herein, an “activator protein” or “activator” with regard togene transcription and/or translation, refers to a protein that binds tooperator of DNA or to RNA to enhance or increase transcription ortranslation, respectively.

As used herein with regard to gene transcription and/or translation,“activation” with regard to gene transcription and/or translation,refers to enhancing or increasing transcription or translation bybinding of activator protein to specific site on DNA or mRNA. In someembodiments, activation includes a significant change in transcriptionor translation level of at least 1.5 fold, in some embodiments at leasttwo fold, and in some embodiments at least five fold.

In certain embodiments, conditions that increase one or more cellularDNA repair processes may include one or more of: introduction of one ormore sites into the GRON or into the plant cell DNA that are targets forbase excision repair, introduction of one or more sites into the GRON orinto the plant cell DNA that are targets for non-homologous end joining,introduction of one or more sites into the GRON or into the plant cellDNA that are targets for microhomology-mediated end joining,introduction of one or more sites into the GRON or into the plant cellDNA that are targets for homologous recombination, and introduction ofone or more sites into the GRON or into the plant cell DNA that aretargets for effecting repair (e.g., base-excision repair (BER);homologous recombination repair (HR); mismatch repair (MMR);non-homologous end-joining repair (NHEJ) which include classical andalternative NHEJ; and nucleotide excision repair (NER)).

As described herein, GRONs for use herein may include one or more of thefollowing alterations from conventional RNA and DNA nucleotides:

-   -   one or more abasic nucleotides;    -   one or more 8′oxo dA and/or 8′oxo dG nucleotides;    -   a reverse base at the 3′ end thereof;    -   one or more 2′O-methyl nucleotides;    -   one or more RNA nucleotides; one or more RNA nucleotides at the        5′ end thereof, and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9,        10, or more; wherein one or more of the RNA nucleotides may        further be modified; one or more RNA nucleotides at the 3′ end        thereof, and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or        more; wherein one or more of the RNA nucleotides may further be        modified;    -   one or more 2′O-methyl RNA nucleotides at the 5′ end thereof,        and in some embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10, or more;    -   an intercalating dye;    -   a 5′ terminus cap;    -   a backbone modification selected from the group consisting of a        phosphothioate modification, a methyl phosphonate modification,        a locked nucleic acid (LNA) modification, a 0 -(2-methoxyethyl)        (MOE) modification, a di PS modification, and a peptide nucleic        acid (PNA) modification;    -   one or more intrastrand crosslinks;    -   one or more fluorescent dyes conjugated thereto, and in some        embodiments at the 5′ or 3′ end of the GRON; and    -   one or more bases which increase hybridization energy.    -   This list is not meant to be limiting.

The term “wobble base” as used herein refers to a change in a one ormore nucleotide bases of a reference nucleotide sequence wherein thechange does not change the sequence of the amino acid coded by thenucleotide relative to the reference sequence.

The term “non-nucleotide” or “abasic nucleotide” as use herein refers toany group or compound which can be incorporated into a nucleic acidchain in the place of one or more nucleotide units, including eithersugar and/or phosphate substitutions, and allows the remaining bases toexhibit their enzymatic activity. The group or compound is abasic inthat it does not contain a commonly recognized nucleotide base, such asadenosine, guanine, cytosine, uracil or thymine. It may havesubstitutions for a 2′ or 3′ H or OH as described in the art and herein.

As described herein, in certain embodiments GRON quality and conversionefficiency may be improved by synthesizing all or a portion of the GRONusing nucleotide multimers, such as dimers, trimers, tetramers, etc.improving its purity.

In certain embodiments, the target deoxyribonucleic acid (DNA) sequenceis within a plant cell, for example the target DNA sequence is in theplant cell genome. The plant cell may be non-transgenic or transgenic,and the target DNA sequence may be a transgene or an endogenous gene ofthe plant cell.

In certain embodiments, the conditions that increase one or morecellular DNA repair processes comprise introducing one or more compoundswhich induce single or double DNA strand breaks into the plant cellprior to, or coincident to, or after delivering the GRON into the plantcell. Exemplary compounds are described herein.

The methods and compositions described herein are applicable to plantsgenerally. By way of example only, a plant species may be selected fromthe group consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat (including but not limited to Triticum spp.,Triticum aestivum, Triticum durum Triticum timopheevii, Triticummonococcum, Triticum spelta, Triticum zhukovskyi and Triticum urartu andhybrids thereof), barley (including but not limited to Hordeum vulgareL., Hordeum comosum, Hordeum depressum, Hordeum intercedens, Hordeumjubatum, Hordeum marinum, Hordeum marinum, Hordeum parodii, Hordeumpusillum, Hordeum secalinum, and Hordeum spontaneum), rice (includingbut not limited to Oryza sativa subsp. indica, Oryza sativa subsp.japonica, Oryza sativa subsp. javanica, Oryza sativa subsp. glutinosa(glutinous rice), Oryza sativa Aromatica group (e.g., basmati), andOryza sativa (floating rice group)), alfalfa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, cassava, potato,carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea,field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye (including but not limited to Secale sylvestre,Secale strictum, Secale cereale, Secale vavilovii, Secale africanum,Secale ciliatoglume, Secale ancestrale, and Secale montanum), oats, turf(including but not limited to Turf grass include Zoysia japonica,Agrostris palustris, Poa pratensis, Poa annua, Digitaria sanguinalis,Cyperus rotundus, Kyllinga brevifolia, Cyperus amuricus, Erigeroncanadensis, Hydrocotyle sibthorpioides, Kummerowia striata, Euphorbiahumifusa, and Viola arvensis) and forage grasses, flax, oilseed rape,cotton, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily,nut-producing plants insofar as they are not already specificallymentioned. These may also apply in whole or in part to all otherbiological systems including but not limited to bacteria, yeast, fungi,algae, and mammalian cells and even their organelles (e.g., mitochondriaand chloroplasts). In some embodiments, the organism or cell is of aspecies selected from the group consisting of Escherichia coli,Mycobacterium smegmatis, Baccillus subtilis, Chlorella, Bacillusthuringiensis, Saccharomyces cerevisiae, Yarrowia lipolytica,Chlamydamonas rhienhardtii, Pichia pastoris, Corynebacterium,Aspergillus niger, and Neurospora crassa. In some embodiments, the yeastis Yarrowia lypolitica. In other embodiments, the yeast is notSaccharomyces cerevisiae. In some embodiments, the plant or plant cellis of a species selected from the group consisting of Arabidopsisthaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Glycine max,Amaranthus tuberculatus, Linum usitatissimum, and Zea mays. The plantspecies may be selected from the group consisting of monocotyledonousplants of the grass family Poaceae. The family Poaceae may be dividedinto two major clades, the clade containing the subfamiliesBambusoideae, Ehrhartoideae, and Pooideae (the BEP clade) and the cladecontaining the subfamilies Panicoideae, Arundinoideae, Chloridoideae,Centothecoideae, Micrairoideae, Aristidoideae, and Danthonioideae (thePACCMAD clade). The subfamily Bambusoideae includes tribe Oryzeae. Theplant species may relate to plants of the BEP clade, in particularplants of the subfamilies Bambusoideae and Ehrhartoideae. The BET cladeincludes subfamilies Bambusoideae, Ehrhartoideae, and group Triticodaeand no other subfamily Pooideae groups. BET crop plants are plants grownfor food or forage that are members of BET subclade, for example barley,corn, etc.

In certain embodiments, the methods further comprise regenerating aplant having a mutation introduced by the GRON from the plant cell, andmay comprise collecting seeds from the plant.

In related aspects, the present disclosure relates to plant cellscomprising a genomic modification introduced by a GRON according to themethods described herein, a plant comprising a genomic modificationintroduced by a GRON according to the methods described herein, or aseed comprising a genomic modification introduced by a GRON according tothe methods described herein; or progeny of a seed comprising a genomicmodification introduced by a GRON according to the methods describedherein.

Other embodiments of the disclosure will be apparent from the followingdetailed description, exemplary embodiments, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts BFP to GFP conversion mediated by phosphothioate (PS)labeled GRONs (having 3 PS moieties at each end of the GRON) and5′Cy3/3′idC labeled GRONs.

FIG. 2A depicts GRONs (SEQ ID NOS 31, 231, 29, and 232, respectively, inorder of appearance) comprising RNA/DNA, referred to herein as“2′-O-methyl GRONs.” FIG. 2B depicts GRONs (SEQ ID NOS 31, 231, 29, and232, respectively, in order of appearance) comprising RNA/DNA, referredto herein as “2′-O-methyl GRONs.”.

FIG. 3 is a schematic of the location on the bfp gene where the BFP5CRISPRs target. Figure discloses SEQ ID NO: 253.

FIG. 4 shows the results of the effect of CRISPRs introduced with eitherthe Cy3 or 3PS GRONs at various lengths, on the percentage of BFP to GFPconversion in a BFP transgenic Arabidopsis thaliana model system.

FIG. 5 shows the results of the effect of CRISPRs introduced with the3PS GRONs at various lengths, on the percentage of BFP to GFP conversionin a BFP transgenic Arabidopsis thaliana model system.

FIG. 6A discloses GRON “gcugcccgug” (SEQ ID NO: 233) used in Example 9.FIG. 6B discloses GRON “gggcgagggc” (SEQ ID NO: 234) used in Example 9.

FIG. 7 shows the measurement of mean percentage GFP positive protoplastsfrom an Arabidopsis thaliana BFP transgenic model system as determinedby flow cytometry from 71-mer GRONs.

FIG. 8 shows the measurement of GFP positive protoplasts fromArabidopsis thaliana BFP transgenic model system as determined by flowcytometry from 201-mer GRONs.

FIG. 9 shows the effect of CRISPRs introduced with coding and non-codingGRONs on the mean percentage of GFP positive cells in a BFP transgenicArabidopsis thaliana model system.

FIG. 10 is a schematic of tethering a single stranded GRON or doublestranded DNA to the CRISPR/Cas complex. Figure discloses SEQ ID NOS235-239, respectively, in order of appearance.

FIG. 11 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of spacers of differing lengths.

FIG. 12 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of spacers were encoded on a plasmid (gRNA plasmid) or usedas an amplicon (gRNA amplicon).

FIG. 13 shows the results of the effect of CRISPRs and GRONs inmediating BFP to GFP conversion in a BFP transgenic Arabidopsis thalianamodel system of unmodified vs. 3PS modified 41-mer GRONs.

FIG. 14 shows the results of next generation sequencing of 3- and 6-weekold Linum usitatissimum (flax) microcalli derived from shoot tipprotoplasts PEG treated with CRISPR plasmid at T=0.

FIG. 15 shows the results of next generation sequencing of 3- and 6-weekold Linum usitatissimum microcalli derived from shoot tip protoplastsPEG treated with CRISPR plasmid at T=0.

FIG. 16A shows the distribution of indels based on size as determined bydeep sequencing in protoplasts treated with CRISPR-Cas plasmid (BC-1) at72 h post delivery. Indels represented 0.79% of the total reads. FIG.16B shows BFP to GFP editing measured by the percentage of GFPfluorescing protoplasts identified by flow cytometry 72 h post deliveryof plasmid (BC-1) and GRON (CG-6). Represented data is not normalizedfor transfection efficiency. Error bars are s.e.m. (n=9).

FIG. 17A shows a comparison of 3PS and unmodified GRONs in BFP to GFPgene editing as measured by flow cytometry at 72 h after delivery ofplasmid (BC-1) and GRONs (CG-1) or (CG-2). FIG. 17B shows a comparisonof GRON lengths in BFP to GFP gene editing as measured by flow cytometryat 72 hours post delivery of plasmid (BC-2) and GRONs (CG-5) or (CG-8).FIG. 17C shows a comparison of3PS to 2′-O-Me GRONs for BFP to GFP geneediting as measured by flow cytometry at 72 h post delivery of plasmid(BC-1) and GRONs (CG-6), (CG-9) or (CG-10). FIG. 17D shows a comparisonof 3PS- to Cy3GRONs in BFP to GFP gene editing as measured by flowcytometry at 72 h post delivery of plasmid (BC-3) and GRONs (CG-3) or(CG-4). Error bars are s.e.m. (n=3). (CG-1): BFP antisense 41 nbunmodified; (CG-2): BFP antisense 41 nb 3PS modified; (CG-3): BFP sense41 nb 3PS modified; (CG-4): BFP sense 41 nb Cy3 modified; (CG-5): BFPsense 60 nb 3PS modified; (CG-6): BFP antisense 201 nb 3PS modified;(CG-8): BFP sense 201 nb 3PS modified; (CG-9): BFP antisense 201 nb2′-O-Me modification on the first 5′ RNA base; (CG-10): BFP antisense201 nb 2′-O-Me modifications on the first nine 5′ RNA bases.

FIG. 18A shows a distribution of indels based on size as determined bydeep sequencing in Arabidopsis protoplasts treated with TALEN plasmid(BT-1) at 72 h post delivery. Indels represented 0.51% of the totalreads. FIG. 18B shows BFP to GFP gene editing as measured by flowcytometry at 48 h post delivery of plasmid (BT-1) and GRON (CG-7). FIG.18C shows a representative distribution of indels based on bp length inL. usitatissimum protoplasts treated with a TALEN (LuET-1) targeting theEPSPS genes 7 d after delivery. Total frequency of indels is 0.50%. FIG.18D shows L. usitatissimum EPSPS gene editing as measured by deepsequencing at 7 d post delivery of plasmid (LuET-1) and GRON (CG-11)into protoplasts. Percentage of total reads represents the number ofreads containing both T97I and P101A edits as a percentage of the totalreads. Error bars are s.e.m. (n=3). (CG-7): BFP sense 201 nb 3PSmodified; (CG-11): EPSPS sense 144 nb Cy3 modified.

FIG. 19 shows effects of the double strand break inducing antibioticszeocin and phleomycin on BFP to GFP editing in transgenic A. thalianaprotoplasts. Protoplasts were treated with zeocin or phleomycin for 90min before PEG introduction of GRON (CG2). Successful editing resultedin GFP fluorescence. Green fluorescing protoplasts were quantified usingan Attune Acoustic Focusing Cytometer.

FIG. 20 shows converted BFP transgenic A. thaliana cells five days afterGRON delivery into BFP transgenic protoplasts, targeting the conversionfrom BFP to GFP. Green fluorescence is indicative of BFP-GFP editing. Abrightfield image; B, the same field of view in blue light. Error barsare s.e.m. (n=4); (CG2): BFP antisense 41 nb 3PS modified; (CG12) BFPantisense 41 nb 3PS modified non-targeting. Images were acquired with anImageXpress Micro system (Molecular Devices, Sunnyvale, Calif., USA)Scale bar=20 μm

FIG. 21A shows a schematic of the CRISPR-Cas plasmid. The mannopinesynthase (Mas) promoter is driving the transcription of the Cas9 genethat is codon optimized for higher plants. The Cas9 gene contains twoSV40 nuclear localization signals (NLS) at either end of the gene and a2X FLAG epitope tag. A. thaliana U6 promoter is driving thetranscription of the gRNA scaffold and transcription is terminated usinga poly(T) signal. FIG. 21B shows a schematic of the TALEN plasmid. TheMas promoter is driving the transcription of the right and left talearms linked together with a 2A ribosome skipping sequence. A Fok1endonuclease is linked to the 3′ end of each Tale arm. The 5′ end of theleft tale contains a nuclear localization signal (NLS) and a V5 epitopetag. rbcT is the Pisum sativum RBCSE9 gene terminator.

FIG. 22A shows a BFP gene target region for the CRISPR-Cas protospacers,BC-1, BC-2 and BC-3 and the TALEN BT-1, left and right tale arms. ThePAM sequence is shown in red. TALEN binding sites are bold andunderlined. The site of BFP to GFP editing CAC→TAC (H66Y) is in boldgreen. FIG. 22B shows an EPSPS gene target region for the TALEN, LuET-1,left and right tale arms. The site of EPSPS conversions ACA>ATA andCCG>GCG (T97I and P101A) are in green. Figure discloses SEQ ID NOS240-245, respectively, in order of appearance.

FIG. 23 shows the amino acid sequence of Alopecurus myosuroides(blackgrass) ACCase gene product (SEQ ID NO:1).

FIG. 24 shows the amino acid sequence of Escherichia coli EPSPS geneproduct (SEQ ID NO:2).

FIG. 25 shows exemplary analogous EPSPS positions.

FIG. 26A (SEQ ID NO: 246) shows an Alopecurus myosuroides plastidalACCase cDNA sequence. FIG. 26B (SEQ ID NO: 247) shows an Alopecurusmyosuroides plastidal ACCase amino acid sequence. FIG. 26C (SEQ ID NO:248) shows an Oryza sativa plastidal ACCase cDNA sequence. FIG. 26D (SEQID NO: 249) shows an Oryza sativa plastidal ACCase amino acid sequence.FIG. 26E (SEQ ID NO: 250) shows an Oryza sativa plastidal ACCase genomicDNA sequence. FIG. 26F (SEQ ID NO: 251) shows an Oryza sativa plastidalACCase protein sequence. FIG. 26G (SEQ ID NO: 252) shows an Oryza sativaACCase protein sequence.

DETAILED DESCRIPTION

Targeted genetic modification mediated by oligonucleotides is a valuabletechnique for use in the specific alteration of short stretches of DNAto create deletions, short insertions, and point mutations. Thesemethods involve DNA pairing/annealing, followed by a DNArepair/recombination event. First, the nucleic acid anneals with itscomplementary strand in the double-stranded DNA in a process mediated bycellular protein factors. This annealing creates a centrally locatedmismatched base pair (in the case of a point mutation), resulting in astructural perturbation that most likely stimulates the endogenousprotein machinery to initiate the second step in the repair process:site-specific modification of the chromosomal sequence and/or that inorganelles (e.g., mitochondria and chloroplasts). This newly introducedmismatch induces the DNA repair machinery to perform a second repairevent, leading to the final revision of the target site. The presentmethods and compositions in various aspects and embodiments disclosedherein, may improve the methods by providing novel approaches whichincrease the availability of DNA repair components, thus increasing theefficiency and reproducibility of gene repair-mediated modifications totargeted nucleic acids.

Efficient methods for site-directed genomic modifications are desirablefor research, clinical gene therapy, industrial microbiology andagriculture. One approach utilizes triplex-forming oligonucleotides(TFO) which bind as third strands to duplex DNA in a sequence-specificmanner, to mediate directed mutagenesis. Such TFO can act either bydelivering a tethered mutagen, such as psoralen or chlorambucil (Havreet al., Proc Nat'l Acad. Sci, U.S.A. 90:7879-7883, 1993; Havre et al., JVirol 67:7323-7331, 1993; Wang et al., Mol Cell Biol 15:1759-1768, 1995;Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991;Belousov et al., Nucleic Acids Res 25:3440-3444, 1997), or by bindingwith sufficient affinity to provoke error-prone repair (Wang et al.,Science 271:802-805, 1996).

Another strategy for genomic modification involves the induction ofhomologous recombination between an exogenous DNA fragment and thetargeted gene. This approach has been used successfully to target anddisrupt selected genes in mammalian cells and has enabled the productionof transgenic mice carrying specific gene knockouts (Capeechi et al.,Science 244:1288-1292, 1989; Wagner, U.S. Pat. No. 4,873,191). Thisapproach involves the transfer of selectable markers to allow isolationof the desired recombinants. Without selection, the ratio of homologousto non-homologous integration of transfected DNA in typical genetransfer experiments is low, usually in the range of 1:1000 or less(Sedivy et al., Gene Targeting, W. H. Freeman and Co., New York, 1992).This low efficiency of homologous integration limits the utility of genetransfer for experimental use or gene therapy. The frequency ofhomologous recombination can be enhanced by damage to the target sitefrom UV irradiation and selected carcinogens (Wang et al., Mol Cell Biol8:196-202, 1988) as well as by site-specific endonucleases (Sedivy etal, Gene Targeting, W. H. Freeman and Co., New York, 1992; Rouet et al.,Proc Nat'l Acad Sci, U.S.A. 91:6064-6068, 1994; Segal et al., Proc Nat'lAcad Sci, U.S.A. 92:806-810, 1995). In addition, DNA damage induced bytriplex-directed psoralen photoadducts can stimulate recombinationwithin and between extrachromosomal vectors (Segal et al., Proc Nat'lAcad Sci, U.S.A. 92:806-810, 1995; Faruqi et al., Mol Cell Biol16:6820-6828, 1996; Glazer, U.S. Pat. No. 5,962,426).

Linear donor fragments are more reconibinogenic than their circularcounterparts (Folger et al., Mol Cell Biol 2:1372-1387, 1982).Recombination can in certain embodiments also be influenced by thelength of uninterrupted homology between both the donor and targetsites, with short fragments often appearing to be ineffective substratesfor recombination (Rubnitz et al., Mol Cell Biol. 4:2253-2258, 1984).Nonetheless, the use of short fragments of DNA or DNA/RNA hybrids forgene correction is the focus of various strategies. (Kunzemann et al.,Gene Ther 3:859-867, 1990.

“Nucleic acid sequence,” “nucleotide sequence” and “polynucleotidesequence” as used herein refer to an oligonucleotide or polynucleotide,and fragments or portions thereof, and to DNA or RNA of genomic orsynthetic origin which may be single- or double-stranded, and representthe sense or antisense strand.

As used herein, the terms “oligonucleotide” and “oligomer” refer to apolymer of nucleobases of at least about 10 nucleobases and as many asabout 1000 nucleobases.

The terms “DNA-modifying molecule” and “DNA-modifying reagent” as usedherein refer to a molecule which is capable of recognizing andspecifically binding to a nucleic acid sequence in the genome of a cell,and which is capable of modifying a target nucleotide sequence withinthe genome, wherein the recognition and specific binding of theDNA-modifying molecule to the nucleic acid sequence isprotein-independent. The term “protein-independent” as used herein inconnection with a DNA-modifying molecule means that the DNA-modifyingmolecule does not require the presence and/or activity of a proteinand/or enzyme for the recognition of, and/or specific binding to, anucleic acid sequence. DNA-modifying molecules are exemplified, but notlimited to triplex forming oligonucleotides, peptide nucleic acids,polyamides, and oligonucleotides which are intended to promote geneconversion. The DNA-modifying molecules of the present disclosure are incertain embodiments distinguished from the prior art's nucleic acidsequences which are used for homologous recombination (Wong & Capecchi,Molec. Cell. Biol. 7:2294-2295, 1987) in that the prior art's nucleicacid sequences which are used for homologous recombination areprotein-dependent. The term “protein-dependent” as used herein inconnection with a molecule means that the molecule requires the presenceand/or activity of a protein and/or enzyme for the recognition of,and/or specific binding of the molecule to, a nucleic acid sequence.Methods for determining whether a DNA-modifying molecule requires thepresence and/or activity of a protein and/or enzyme for the recognitionof, and/or specific binding to, a nucleic acid sequence are within theskill in the art (see, e.g., Dennis et al. Nucl. Acids Res.27:4734-4742, 1999). For example, the DNA-modifying molecule may beincubated in vitro with the nucleic acid sequence in the absence of anyproteins and/or enzymes. The detection of specific binding between theDNA-modifying molecule and the nucleic acid sequence demonstrates thatthe DNA-modifying molecule is protein-independent. On the other hand,the absence of specific binding between the DNA-modifying molecule andthe nucleic acid sequence demonstrates that the DNA-modifying moleculeis protein-dependent and/or requires additional factors.

“Triplex forming oligonucleotide” (TFO) is defined as a sequence of DNAor RNA that is capable of binding in the major grove of a duplex DNA orRNA helix to form a triple helix. Although the TFO is not limited to anyparticular length, a preferred length of the TFO is 250 nucleotides orless, 200 nucleotides or less, or100 nucleotides or less, or from 5 to50 nucleotides, or from 10 to 25 nucleotides, or from 15 to 25nucleotides. Although a degree of sequence specificity between the TFOand the duplex DNA is necessary for formation of the triple helix, noparticular degree of specificity is required, as long as the triplehelix is capable of forming. Likewise, no specific degree of avidity oraffinity between the TFO and the duplex helix is required as long as thetriple helix is capable of forming. While not intending to limit thelength of the nucleotide sequence to which the TFO specifically binds inone embodiment, the nucleotide sequence to which the TFO specificallybinds is from 1 to 100, in some embodiments from 5 to 50, yet otherembodiments from 10 to 25, and in other embodiments from 15 to 25,nucleotides. Additionally, “triple helix” is defined as a double-helicalnucleic acid with an oligonucleotide bound to a target sequence withinthe double-helical nucleic acid. The “double-helical” nucleic acid canbe any double-stranded nucleic acid including double-stranded DNA,double-stranded RNA and mixed duplexes of DNA and RNA. Thedouble-stranded nucleic acid is not limited to any particular length.However, in preferred embodiments it has a length of greater than 500bp, in some embodiments greater than 1 kb and in some embodimentsgreater than about 5 kb. In many applications the double-helical nucleicacid is cellular, genomic nucleic acid. The triplex formingoligonucleotide may bind to the target sequence in a parallel oranti-parallel manner.

“Peptide Nucleic Acids,” “polyamides” or “PNA” are nucleic acids whereinthe phosphate backbone is replaced with an N-aminoethylglycine-basedpolyamide structure. PNAs have a higher affinity for complementarynucleic acids than their natural counter parts following theWatson-Crick base-pairing rules. PNAs can form highly stable triplehelix structures with DNA of the following stoichiometry: (PNA)2.DNA.Although the peptide nucleic acids and polyamides are not limited to anyparticular length, a preferred length of the peptide nucleic acids andpolyamides is 200 nucleotides or less, in some embodiments 100nucleotides or less, and in some embodiments from 5 to 50 nucleotideslong. While not intending to limit the length of the nucleotide sequenceto which the peptide nucleic acid and polyamide specifically binds, inone embodiment, the nucleotide sequence to which the peptide nucleicacid and polyamide specifically bind is from 1 to 100, in someembodiments from 5 to 50, yet other embodiments from 5 to 25, and otherembodiments from 5 to 20, nucleotides.

The term “cell” refers to a single cell. The term “cells” refers to apopulation of cells. The population may be a pure population comprisingone cell type. Likewise, the population may comprise more than one celltype. In the present disclosure, there is no limit on the number of celltypes that a cell population may comprise. A cell as used hereinincludes without limitation plant callus cells, cells with and withoutcell walls, prokaryotic cells and eukaryotic cells.

The term “synchronize” or “synchronized,” when referring to a sample ofcells, or “synchronized cells” or “synchronized cell population” refersto a plurality of cells which have been treated to cause the populationof cells to be in the same phase of the cell cycle. It is not necessarythat all of the cells in the sample be synchronized. A small percentageof cells may not be synchronized with the majority of the cells in thesample. A preferred range of cells that are synchronized is between10-100%. A more preferred range is between 30-100%. Also, it is notnecessary that the cells be a pure population of a single cell type.More than one cell type may be contained in the sample. In this regard,only one of cell types may be synchronized or may be in a differentphase of the cell cycle as compared to another cell type in the sample.

The term “synchronized cell” when made in reference to a single cellmeans that the cell has been manipulated such that it is at a cell cyclephase which is different from the cell cycle phase of the cell prior tothe manipulation. Alternatively, a “synchronized cell” refers to a cellthat has been manipulated to alter (i.e., increase or decrease) theduration of the cell cycle phase at which the cell was prior to themanipulation when compared to a control cell (e.g., a cell in theabsence of the manipulation).

The term “cell cycle” refers to the physiological and morphologicalprogression of changes that cells undergo when dividing (i.e.proliferating). The cell cycle is generally recognized to be composed ofphases termed “interphase,” “prophase,” “metaphase,” “anaphase,” and“telophase”. Additionally, parts of the cell cycle may be termed “M(mitosis),” “S (synthesis),” “G0,” “G1 (gap 1)” and “G2 (gap2)”.Furthermore, the cell cycle includes periods of progression that areintermediate to the above named phases.

The term “cell cycle inhibition” refers to the cessation of cell cycleprogression in a cell or population of cells. Cell cycle inhibition isusually induced by exposure of the cells to an agent (chemical,proteinaceous or otherwise) that interferes with aspects of cellphysiology to prevent continuation of the cell cycle.

“Proliferation” or “cell growth” refers to the ability of a parent cellto divide into two daughter cells repeatably thereby resulting in atotal increase of cells in the population. The cell population may be inan organism or in a culture apparatus.

The term “capable of modifying DNA” or “DNA modifying means” refers toprocedures, as well as endogenous or exogenous agents or reagents thathave the ability to induce, or can aid in the induction of, changes tothe nucleotide sequence of a targeted segment of DNA. Such changes maybe made by the deletion, addition or substitution of one or more baseson the targeted DNA segment. It is not necessary that the DNA sequencechanges confer functional changes to any gene encoded by the targetedsequence. Furthermore, it is not necessary that changes to the DNA bemade to any particular portion or percentage of the cells.

The term “nucleotide sequence of interest” refers to any nucleotidesequence, the manipulation of which may be deemed desirable for anyreason, by one of ordinary skill in the art. Such nucleotide sequencesinclude, but are not limited to, coding sequences of structural genes(e.g., reporter genes, selection marker genes, oncogenes, drugresistance genes, growth factors, etc.), and non-coding regulatorysequences that do not encode an mRNA or protein product (e.g., promotersequence, enhancer sequence, polyadenylation sequence, terminationsequence, regulatory RNAs such as miRNA, etc.).

“Amino acid sequence,” “polypeptide sequence,” “peptide sequence” and“peptide” are used interchangeably herein to refer to a sequence ofamino acids.

“Target sequence,” as used herein, refers to a double-helical nucleicacid comprising a sequence greater than 8 nucleotides in length but lessthan 201 nucleotides in length. In some embodiments, the target sequenceis between 8 to 30 bases. The target sequence, in general, is defined bythe nucleotide sequence on one of the strands on the double-helicalnucleic acid.

As used herein, a “purine-rich sequence” or “polypurine sequence” whenmade in reference to a nucleotide sequence on one of the strands of adouble-helical nucleic acid sequence is defined as a contiguous sequenceof nucleotides wherein greater than 50% of the nucleotides of the targetsequence contain a purine base. However, it is preferred that thepurine-rich target sequence contain greater than 60% purine nucleotides,in some embodiments greater than 75% purine nucleotides, in otherembodiments greater than 90% purine nucleotides and yet otherembodiments 100% purine nucleotides.

As used herein, a “pyrimidine-rich sequence” or “polypyrimidinesequence” when made in reference to a nucleotide sequence on one of thestrands of a double-helical nucleic acid sequence is defined as acontiguous sequence of nucleotides wherein greater that 50% of thenucleotides of the target sequence contain a pyrimidine base. However,it is preferred that the pyrimidine-rich target sequence contain greaterthan 60% pyrimidine nucleotides and in some embodiments greater than 75%pyrimidine nucleotides. In some embodiments, the sequence containsgreater than 90% pyrimidine nucleotides and, in other embodiments, is100% pyrimidine nucleotides.

A “variant” of a first nucleotide sequence is defined as a nucleotidesequence which differs from the first nucleotide sequence (e.g., byhaving one or more deletions, insertions, or substitutions that may bedetected using hybridization assays or using DNA sequencing). Includedwithin this definition is the detection of alterations or modificationsto the genomic sequence of the first nucleotide sequence. For example,hybridization assays may be used to detect (1) alterations in thepattern of restriction enzyme fragments capable of hybridizing to thefirst nucleotide sequence when comprised in a genome (i.e., RFLPanalysis), (2) the inability of a selected portion of the firstnucleotide sequence to hybridize to a sample of genomic DNA whichcontains the first nucleotide sequence (e.g., using allele-specificoligonucleotide probes), (3) improper or unexpected hybridization, suchas hybridization to a locus other than the normal chromosomal locus forthe first nucleotide sequence (e.g., using fluorescent in situhybridization (FISH) to metaphase chromosomes spreads, etc.). Oneexample of a variant is a mutated wild type sequence.

The terms “nucleic acid” and “unmodified nucleic acid” as used hereinrefer to any one of the known four deoxyribonucleic acid bases (i.e.,guanine, adenine, cytosine, and thymine). The term “modified nucleicacid” refers to a nucleic acid whose structure is altered relative tothe structure of the unmodified nucleic acid. Illustrative of suchmodifications would be replacement covalent modifications of the bases,such as alkylation of amino and ring nitrogens as well as saturation ofdouble bonds.

As used herein, the terms “mutation” and “modification” and grammaticalequivalents thereof when used in reference to a nucleic acid sequenceare used interchangeably to refer to a deletion, insertion,substitution, strand break, and/or introduction of an adduct. A“deletion” is defined as a change in a nucleic acid sequence in whichone or more nucleotides is absent. An “insertion” or “addition” is thatchange in a nucleic acid sequence which has resulted in the addition ofone or more nucleotides. A “substitution” results from the replacementof one or more nucleotides by a molecule which is a different moleculefrom the replaced one or more nucleotides. For example, a nucleic acidmay be replaced by a different nucleic acid as exemplified byreplacement of a thymine by a cytosine, adenine, guanine, or uridine.Pyrimidine to pyrimidine (e.g. C to T or T to C nucleotidesubstitutions) or purine to purine (e.g. G to A or A to G nucleotidesubstitutions) are termed transitions, whereas pyrimidine to purine orpurine to pyrimidine (e.g. G to T or G to C or A to T or A to C) aretermed transversions. Alternatively, a nucleic acid may be replaced by amodified nucleic acid as exemplified by replacement of a thymine bythymine glycol. Mutations may result in a mismatch. The term “mismatch”refers to a non-covalent interaction between two nucleic acids, eachnucleic acid residing on a different polynucleic acid sequence, whichdoes not follow the base-pairing rules. For example, for the partiallycomplementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch (atransition) is present. The terms “introduction of an adduct” or “adductformation” refer to the covalent or non-covalent linkage of a moleculeto one or more nucleotides in a DNA sequence such that the linkageresults in a reduction (in some embodiments from 10% to 100%, in otherembodiments from 50% to 100%, and in some embodiments from 75% to 100%)in the level of DNA replication and/or transcription.

The term “DNA cutter” refers to a moiety that effects a strand break.Non-limited examples include meganucleases, TALEs/TALENs, antibiotics,zinc fingers and CRISPRs or CRISPR/cas systems.

The term “strand break” when made in reference to a double strandednucleic acid sequence includes a single-strand break and/or adouble-strand break. A single-strand break (a nick) refers to aninterruption in one of the two strands of the double stranded nucleicacid sequence. This is in contrast to a double-strand break which refersto an interruption in both strands of the double stranded nucleic acidsequence, which may result in blunt or staggered ends. Strand breaks maybe introduced into a double stranded nucleic acid sequence eitherdirectly (e.g., by ionizing radiation or treatment with certainchemicals) or indirectly (e.g., by enzymatic incision at a nucleic acidbase).

The terms “mutant cell” and “modified cell” refer to a cell whichcontains at least one modification in the cell's genomic sequence.

The term “portion” when used in reference to a nucleotide sequencerefers to fragments of that nucleotide sequence. The fragments may rangein size from 5 nucleotide residues to the entire nucleotide sequenceminus one nucleic acid residue.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring. An end of an oligonucleotide is referred toas the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate ofanother mononucleotide pentose ring. As used herein, a nucleic acidsequence, even if internal to a larger oligonucleotide, also may be saidto have 5′ and 3′ ends. In either a linear or circular DNA molecule,discrete elements are referred to as being “upstream” or 5′ of the“downstream” or 3′ elements. This terminology reflects thattranscription proceeds in a 5′ to 3′ direction along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule which is expressed using arecombinant DNA molecule.

As used herein, the terms “vector” and “vehicle” are usedinterchangeably in reference to nucleic acid molecules that transfer DNAsegment(s) from one cell to another.

The terms “in operable combination,” “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The terms also refer to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “transfection” as used herein refers to the introduction offoreign DNA into cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofectin, protoplastfusion, retroviral infection, biolistics (i.e., particle bombardment)and the like.

As used herein, the terms “complementary” or “complementarity” are usedin reference to “polynucleotides” and “oligonucleotides” (which areinterchangeable terms that refer to a sequence of nucleotides) relatedby the base-pairing rules. For example, the sequence “5′-CAGT-3′,” iscomplementary to the sequence “5′-ACTG-3′.” Complementarity can be“partial” or “total”. “Partial” complementarity is where one or morenucleic acid bases is not matched according to the base pairing rules.“Total” or “complete” complementarity between nucleic acids is whereeach and every nucleic acid base is matched with another base under thebase pairing rules. The degree of complementarity between nucleic acidstrands may have significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This may be of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids. For the sake ofconvenience, the terms “polynucleotides” and “oligonucleotides” includemolecules which include nucleosides.

The terms “homology” and “homologous” as used herein in reference tonucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). When used in reference to a double-stranded nucleicacid sequence such as a cDNA or genomic clone, the term “substantiallyhomologous” refers to any nucleic acid sequence (e.g., probe) which canhybridize to either or both strands of the double-stranded nucleic acidsequence under conditions of low stringency as described above. Anucleotide sequence which is partially complementary, i.e.,“substantially homologous,” to a nucleic acid sequence is one that atleast partially inhibits a completely complementary sequence fromhybridizing to a target nucleic acid sequence. The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence to a target sequence under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target.

Low stringency conditions comprise conditions equivalent to binding orhybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent (50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 2.0×SSPE, 0.1% SDS at room temperature when a probe of about100 to about 1000 nucleotides in length is employed.

In addition, conditions which promote hybridization under conditions ofhigh stringency (e.g., increasing the temperature of the hybridizationand/or wash steps, the use of formamide in the hybridization solution,etc.) are well known in the art. High stringency conditions, when usedin reference to nucleic acid hybridization, comprise conditionsequivalent to binding or hybridization at 68° C. in a solutionconsisting of 5×SSPE, 1% SDS, 5×Denhardt′s reagent and 100 μg/mldenatured salmon sperm DNA followed by washing in a solution comprising0.1×SSPE and 0.1% SDS at 68° C. when a probe of about 100 to about 1000nucleotides in length is employed.

It is well known in the art that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol), as well as components of the hybridizationsolution may be varied to generate conditions of low stringencyhybridization different from, but equivalent to, the above listedconditions.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 50% to 70%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 50% to 70% homology to the first nucleicacid sequence.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids using any process by which astrand of nucleic acid joins with a complementary strand through basepairing to form a hybridization complex. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the Tm of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein the term “hybridization complex” refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term “Tm” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization, 1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of Tm.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. “Stringency” typically occurs in a rangefrom about Tm-5° C. (5° C. below the melting temperature of the probe)to about 20° C. to 25° C. below Tm. As will be understood by those ofskill in the art, a stringent hybridization can be used to identify ordetect identical polynucleotide sequences or to identify or detectsimilar or related polynucleotide sequences.

The terms “specific binding,” “binding specificity,” and grammaticalequivalents thereof when made in reference to the binding of a firstnucleotide sequence to a second nucleotide sequence, refer to thepreferential interaction between the first nucleotide sequence with thesecond nucleotide sequence as compared to the interaction between thesecond nucleotide sequence with a third nucleotide sequence. Specificbinding is a relative term that does not require absolute specificity ofbinding; in other words, the term “specific binding” does not requirethat the second nucleotide sequence interact with the first nucleotidesequence in the absence of an interaction between the second nucleotidesequence and the third nucleotide sequence. Rather, it is sufficientthat the level of interaction between the first nucleotide sequence andthe second nucleotide sequence is greater than the level of interactionbetween the second nucleotide sequence with the third nucleotidesequence. “Specific binding” of a first nucleotide sequence with asecond nucleotide sequence also means that the interaction between thefirst nucleotide sequence and the second nucleotide sequence isdependent upon the presence of a particular structure on or within thefirst nucleotide sequence; in other words the second nucleotide sequenceis recognizing and binding to a specific structure on or within thefirst nucleotide sequence rather than to nucleic acids or to nucleotidesequences in general. For example, if a second nucleotide sequence isspecific for structure “A” that is on or within a first nucleotidesequence, the presence of a third nucleic acid sequence containingstructure A will reduce the amount of the second nucleotide sequencewhich is bound to the first nucleotide sequence.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

The terms “heterologous nucleic acid sequence” or “heterologous DNA” areused interchangeably to refer to a nucleotide sequence which is ligatedto a nucleic acid sequence to which it is not ligated in nature, or towhich it is ligated at a different location in nature. Heterologous DNAis not endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchheterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

“Amplification” is defined as the production of additional copies of anucleic acid sequence and is generally carried out using polymerasechain reaction technologies well known in the art (Dieffenbach C W and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y.). As used herein, the term “polymerase chainreaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos.4,683,195, and 4,683,202, hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. The length of the amplified segment of the desired targetsequence is determined by the relative positions of two oligonucleotideprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(“PCR”). Because the desired amplified segments of the target sequencebecome the predominant sequences (in terms of concentration) in themixture, they are said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications.

One such preferred method, particularly for commercial applications, isbased on the widely used TaqMan® real-time PCR technology, and combinesAllele-Specific PCR with a Blocking reagent (ASB-PCR) to suppressamplification of the wildtype allele. ASB-PCR can be used for detectionof germ line or somatic mutations in either DNA or RNA extracted fromany type of tissue, including formalin-fixed paraffin-embedded tumorspecimens. A set of reagent design rules are developed enablingsensitive and selective detection of single point substitutions,insertions, or deletions against a background of wild-type allele inthousand-fold or greater excess. (Morlan J, Baker J, Sinicropi DMutation Detection by Real-Time PCR: A Simple, Robust and HighlySelective Method. PLoS ONE 4(2): e4584, 2009)

The terms “reverse transcription polymerase chain reaction” and “RT-PCR”refer to a method for reverse transcription of an RNA sequence togenerate a mixture of cDNA sequences, followed by increasing theconcentration of a desired segment of the transcribed cDNA sequences inthe mixture without cloning or purification. Typically, RNA is reversetranscribed using a single primer (e.g., an oligo-dT primer) prior toPCR amplification of the desired segment of the transcribed DNA usingtwo primers.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and of an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). In someembodiments, the primer is single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. In some embodiments, theprimer is an oligodeoxyribonucleotide. The primer must be sufficientlylong to prime the synthesis of extension products in the presence of theinducing agent. The exact lengths of the primers will depend on manyfactors, including temperature, source of primer and the use of themethod.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present disclosure will be labeled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present disclosure be limited toany particular detection system or label.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut or nick double-or single-stranded DNA at or near a specific nucleotide sequence, forexample, an endonuclease domain of a type IIS restriction endonuclease(e.g., FokI can be used, as taught by Kim et al., 1996, Proc. Nat'l.Acad. Sci. USA, 6:1 156-60).

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene, i.e. the nucleic acid sequence which encodes agene product. The coding region may be present in either a cDNA, genomicDNA or RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded.Additionally “an oligonucleotide having a nucleotide sequence encoding agene” may include suitable control elements such as enhancers,promoters, splice junctions, polyadenylation signals, etc. if needed topermit proper initiation of transcription and/or correct processing ofthe primary RNA transcript. Further still, the coding region of thepresent disclosure may contain endogenous enhancers, splice junctions,intervening sequences, polyadenylation signals, etc.

Transcriptional control signals in eukaryotes comprise “enhancer”elements. Enhancers consist of short arrays of DNA sequences thatinteract specifically with cellular proteins involved in transcription(Maniatis, T. et al., Science 236:1237, 1987). Enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inplant, yeast, insect and mammalian cells and viruses. The selection of aparticular enhancer depends on what cell type is to be used to expressthe protein of interest.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York, pp. 16.7-16.8, 1989). A commonly used splicedonor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene.

The term “promoter,” “promoter element” or “promoter sequence” as usedherein, refers to a DNA sequence which when placed at the 5′ end of(i.e., precedes) an oligonucleotide sequence is capable of controllingthe transcription of the oligonucleotide sequence into mRNA. A promoteris typically located 5′ (i.e., upstream) of an oligonucleotide sequencewhose transcription into mRNA it controls, and provides a site forspecific binding by RNA polymerase and for initiation of transcription.

The term “promoter activity” when made in reference to a nucleic acidsequence refers to the ability of the nucleic acid sequence to initiatetranscription of an oligonucleotide sequence into mRNA.

The term “tissue specific” as it applies to a promoter refers to apromoter that is capable of directing selective expression of anoligonucleotide sequence to a specific type of tissue in the relativeabsence of expression of the same oligonucleotide in a different type oftissue. Tissue specificity of a promoter may be evaluated by, forexample, operably linking a reporter gene to the promoter sequence togenerate a reporter construct, introducing the reporter construct intothe genome of a plant or an animal such that the reporter construct isintegrated into every tissue of the resulting transgenic animal, anddetecting the expression of the reporter gene (e.g., detecting mRNA,protein, or the activity of a protein encoded by the reporter gene) indifferent tissues of the transgenic plant or animal. Selectivity neednot be absolute. The detection of a greater level of expression of thereporter gene in one or more tissues relative to the level of expressionof the reporter gene in other tissues shows that the promoter isspecific for the tissues in which greater levels of expression aredetected.

The term “cell type specific” as applied to a promoter refers to apromoter which is capable of directing selective expression of anoligonucleotide sequence in a specific type of cell in the relativeabsence of expression of the same oligonucleotide sequence in adifferent type of cell within the same tissue. The term “cell typespecific” when applied to a promoter also means a promoter capable ofpromoting selective expression of an oligonucleotide in a region withina single tissue. Again, selectivity need not be absolute. Cell typespecificity of a promoter may be assessed using methods well known inthe art, e.g., immunohistochemical staining as described herein.Briefly, tissue sections are embedded in paraffin, and paraffin sectionsare reacted with a primary antibody which is specific for thepolypeptide product encoded by the oligonucleotide sequence whoseexpression is controlled by the promoter. As an alternative to paraffinsectioning, samples may be cryosectioned. For example, sections may befrozen prior to and during sectioning thus avoiding potentialinterference by residual paraffin. A labeled (e.g., peroxidaseconjugated) secondary antibody which is specific for the primaryantibody is allowed to bind to the sectioned tissue and specific bindingdetected (e.g., with avidin/biotin) by microscopy.

The terms “selective expression,” “selectively express” and grammaticalequivalents thereof refer to a comparison of relative levels ofexpression in two or more regions of interest. For example, “selectiveexpression” when used in connection with tissues refers to asubstantially greater level of expression of a gene of interest in aparticular tissue, or to a substantially greater number of cells whichexpress the gene within that tissue, as compared, respectively, to thelevel of expression of, and the number of cells expressing, the samegene in another tissue (i.e., selectivity need not be absolute).Selective expression does not require, although it may include,expression of a gene of interest in a particular tissue and a totalabsence of expression of the same gene in another tissue. Similarly,“selective expression” as used herein in reference to cell types refersto a substantially greater level of expression of, or a substantiallygreater number of cells which express, a gene of interest in aparticular cell type, when compared, respectively, to the expressionlevels of the gene and to the number of cells expressing the gene inanother cell type.

The term “contiguous” when used in reference to two or more nucleotidesequences means the nucleotide sequences are ligated in tandem either inthe absence of intervening sequences, or in the presence of interveningsequences which do not comprise one or more control elements.

As used herein, the terms “nucleic acid molecule encoding,” “nucleotideencoding,” “DNA sequence encoding” and “DNA encoding” refer to the orderor sequence of deoxyribonucleotides along a strand of deoxyribonucleicacid. The order of these deoxyribonucleotides determines the order ofamino acids along the polypeptide (protein) chain. The DNA sequence thuscodes for the amino acid sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isseparated from at least one contaminant nucleic acid with which it isordinarily associated in its natural source. Isolated nucleic acid isnucleic acid present in a form or setting that is different from that inwhich it is found in nature. In contrast, non-isolated nucleic acids arenucleic acids such as DNA and RNA which are found in the state theyexist in nature. For example, a given DNA sequence (e.g., a gene) isfound on the host cell chromosome in proximity to neighboring genes; RNAsequences, such as a specific mRNA sequence encoding a specific protein,are found in the cell as a mixture with numerous other mRNAs whichencode a multitude of proteins. However, isolated nucleic acid encodinga polypeptide of interest includes, by way of example, such nucleic acidin cells ordinarily expressing the polypeptide of interest where thenucleic acid is in a chromosomal or extrachromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid or oligonucleotide may be present in single-stranded ordouble-stranded form. Isolated nucleic acid can be readily identified(if desired) by a variety of techniques (e.g., hybridization, dotblotting, etc.). When an isolated nucleic acid or oligonucleotide is tobe utilized to express a protein, the oligonucleotide will contain at aminimum the sense or coding strand (i.e., the oligonucleotide may besingle-stranded). Alternatively, it may contain both the sense andanti-sense strands (i.e., the oligonucleotide may be double-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof one or more (undesired) components from a sample. For example, whererecombinant polypeptides are expressed in bacterial host cells, thepolypeptides are purified by the removal of host cell proteins therebyincreasing the percent of recombinant polypeptides in the sample.

As used herein, the term “substantially purified” refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,in some embodiments 75% free and other embodiments 90% free from othercomponents with which they are naturally associated. An “isolatedpolynucleotide” is, therefore, a substantially purified polynucleotide.

As used herein the term “coding region” when used in reference to astructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side generally by the nucleotide triplet “ATG” which encodes theinitiator methionine and on the 3′ side by one of the three tripletswhich specify stop codons (i.e., TAA, TAG, TGA).

By “coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

By “non-coding sequence” is meant a sequence of a nucleic acid or itscomplement, or a part thereof, that is not transcribed into amino acidin vivo, or where tRNA does not interact to place or attempt to place anamino acid. Non-coding sequences include both intron sequences ingenomic DNA or immature primary RNA transcripts, and gene-associatedsequences such as promoters, enhancers, silencers, etc.

As used herein, the term “structural gene” or “structural nucleotidesequence” refers to a DNA sequence coding for RNA or a protein whichdoes not control the expression of other genes. In contrast, a“regulatory gene” or “regulatory sequence” is a structural gene whichencodes products (e.g., transcription factors) which control theexpression of other genes.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the term “peptide transcription factor binding site” or“transcription factor binding site” refers to a nucleotide sequencewhich binds protein transcription factors and, thereby, controls someaspect of the expression of nucleic acid sequences. For example, Sp-1and AP1 (activator protein 1) binding sites are examples of peptidetranscription factor binding sites.

As used herein, the term “gene” means the deoxyribonucleotide sequencescomprising the coding region of a structural gene. A “gene” may alsoinclude non-translated sequences located adjacent to the coding regionon both the 5′ and 3′ ends such that the gene corresponds to the lengthof the full-length mRNA. The sequences which are located 5′ of thecoding region and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into heterogenous nuclearRNA (hnRNA); introns may contain regulatory elements such as enhancers.Introns are removed or “spliced out” from the nuclear or primarytranscript; introns therefore are absent in the messenger RNA (mRNA)transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide. A gene isgenerally a single locus. In a normal diploid organism, a gene has twoalleles. In tetraploid potato, however, each gene has 4 alleles. Insugarcane, which is dodecaploid there can be 12 alleles per gene.Particular examples include flax which has two EPSPS loci each with twoalleles and rice which has a single homomeric plastidal ACCase with twoalleles.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

A “non-human animal” refers to any animal which is not a human andincludes vertebrates such as rodents, non-human primates, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, ayes, etc. Preferred non-human animals are selected from theorder Rodentia. “Non-human animal” additionally refers to amphibians(e.g. Xenopus), reptiles, insects (e.g. Drosophila) and othernon-mammalian animal species.

As used herein, the term “transgenic” refers to an organism or cell thathas DNA derived from another organism inserted into which becomesintegrated into the genome either of somatic and/or germ line cells ofthe plant or animal. A “transgene” means a DNA sequence which is partlyor entirely heterologous (i.e., not present in nature) to the plant oranimal in which it is found, or which is homologous to an endogenoussequence (i.e., a sequence that is found in the animal in nature) and isinserted into the plant' or animal's genome at a location which differsfrom that of the naturally occurring sequence. Transgenic plants oranimals which include one or more transgenes are within the scope ofthis disclosure. Additionally, a “transgenic” as used herein refers toan organism that has had one or more genes modified and/or “knocked out”(made non-functional or made to function at reduced level, i.e., a“knockout” mutation) by the disclosure's methods, by homologousrecombination, TFO mutation or by similar processes. For example, insome embodiments, a transgenic organism or cell includes inserted DNAthat includes a foreign promoter and/or coding region.

A “transformed cell” is a cell or cell line that has acquired theability to grow in cell culture for multiple generations, the ability togrow in soft agar, and/or the ability to not have cell growth inhibitedby cell-to-cell contact. In this regard, transformation refers to theintroduction of foreign genetic material into a cell or organism.Transformation may be accomplished by any method known which permits thesuccessful introduction of nucleic acids into cells and which results inthe expression of the introduced nucleic acid. “Transformation” includesbut is not limited to such methods as transfection, microinjection,electroporation, nucleofection and lipofection (liposome-mediated genetransfer). Transformation may be accomplished through use of anyexpression vector. For example, the use of baculovirus to introduceforeign nucleic acid into insect cells is contemplated. The term“transformation” also includes methods such as P-element mediatedgermline transformation of whole insects. Additionally, transformationrefers to cells that have been transformed naturally, usually throughgenetic mutation.

As used herein “exogenous” means that the gene encoding the protein isnot normally expressed in the cell. Additionally, “exogenous” refers toa gene transfected into a cell to augment the normal (i.e. natural)level of expression of that gene.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (i.e., “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell intowhich it is introduced and which contains some modification relative tothe naturally-occurring sequence. Generally, although not necessarily,heterologous DNA encodes heterologous RNA and heterologous proteins thatare not normally produced by the cell into which it is introduced.Examples of heterologous DNA include reporter genes, transcriptional andtranslational regulatory sequences, DNA sequences which encodeselectable marker proteins (e.g., proteins which confer drugresistance), etc.

Constructs

The nucleic acid molecules disclosed herein (e.g., site specificnucleases, or guide RNA for CRISPRs) can be used in the production ofrecombinant nucleic acid constructs. In one embodiment, the nucleic acidmolecules of the present disclosure can be used in the preparation ofnucleic acid constructs, for example, expression cassettes forexpression in the plant, microorganism, or animal of interest. Thisexpression may be transient for instance when the construct is notintegrated into the host genome or maintained under the control offeredby the promoter and the position of the construct within the host'sgenome if it becomes integrated.

Expression cassettes may include regulatory sequences operably linked tothe site specific nuclease or guide RNA sequences disclosed herein. Thecassette may additionally contain at least one additional gene to beco-transformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes.

The nucleic acid constructs may be provided with a plurality ofrestriction sites for insertion of the site specific nuclease codingsequence to be under the transcriptional regulation of the regulatoryregions. The nucleic acid constructs may additionally contain nucleicacid molecules encoding for selectable marker genes.

Any promoter can be used in the production of the nucleic acidconstructs. The promoter may be native or analogous, or foreign orheterologous, to the plant, microbial, or animal host nucleic acidsequences disclosed herein. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. Where thepromoter is “foreign” or “heterologous” to the plant, microbial, oranimal host, it is intended that the promoter is not found in the nativeplant, microbial, or animal into which the promoter is introduced. Asused herein, a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

The site directed nuclease sequences disclosed herein may be expressedusing heterologous promoters.

Any promoter can be used in the preparation of constructs to control theexpression of the site directed nuclease sequences, such as promotersproviding for constitutive, tissue-preferred, inducible, or otherpromoters for expression in plants, microbes, or animals. Constitutivepromoters include, for example, the core promoter of the Rsyn7 promoterand other constitutive promoters disclosed in WO 99/43 838 and U.S. Pat.No. 6,072,050; the core CaMV 35S promoter (Odell et al. Nature313:810-812; 1985); rice actin (McElroy et al., Plant Cell 2:163-171,1990); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632, 1989and Christensen et al., Plant Mol. Biol. 18:675-689, 1992); pEMU (Lastet al., Theor. Appl. Genet. 81:581-588, 1991); MAS (Velten et al., EMBOJ. 3:2723-2730, 1984); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, U.S. PatentNos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to direct site directednuclease expression within a particular plant tissue. Suchtissue-preferred promoters include, but are not limited to,leaf-preferred promoters, root-preferred promoters, seed-preferredpromoters, and stem-preferred promoters. Tissue-preferred promotersinclude Yamamoto et al., Plant J. 12(2):255-265, 1997; Kawamata et al.,Plant Cell Physiol. 38(7):792-803, 1997; Hansen et al., Mol. Gen Genet.254(3):337-343, 1997; Russell et al., Transgenic Res. 6(2):157-168,1997; Rinehart et al., Plant Physiol. 1 12(3):1331-1341, 1996; Van Campet al., Plant Physiol. 1 12(2):525-535, 1996; Canevascini et al., PlantPhysiol. 112(2): 513-524, 1996; Yamamoto et al., Plant Cell Physiol.35(5):773-778, 1994; Lam, Results Probl. Cell Differ. 20:181-196, 1994;Orozco et al. Plant Mol Biol. 23(6):1129-1138, 1993; Matsuoka et al.,Proc Nat'l. Acad. Sci. USA 90(20):9586-9590, 1993; and Guevara-Garcia etal., Plant J. 4(3):495-505, 1993.

The nucleic acid constructs may also include transcription terminationregions. Where transcription terminations regions are used, anytermination region may be used in the preparation of the nucleic acidconstructs. For example, the termination region may be derived fromanother source (i.e., foreign or heterologous to the promoter). Examplesof termination regions that are available for use in the constructs ofthe present disclosure include those from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al., Mol. Gen. Genet.262:141-144, 1991; Proudfoot, Cell 64:671-674, 1991; Sanfacon et al.,Genes Dev. 5:141-149, 1991; Mogen et al., Plant Cell 2:1261-1272, 1990;Munroe et al., Gene 91:151-158, 1990; Ballas et al., Nucleic Acids Res.17:7891-7903, 1989; and Joshi et al., Nucleic Acid Res. 15:9627-9639,1987.

In conjunction with any of the aspects, embodiments, methods and/orcompositions disclosed herein, the nucleic acids may be optimized forincreased expression in the transformed plant. That is, the nucleicacids encoding the site directed nuclease proteins can be synthesizedusing plant-preferred codons for improved expression. See, for example,Campbell and Gowri, (Plant Physiol. 92:1-11, 1990) for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, and 5,436,391, and Murray et al., Nucleic Acids Res.17:477-498, 1989. See also e.g., Lanza et al., BMC Systems Biology8:33-43, 2014; Burgess-Brown et al., Protein Expr. Purif. 59:94-102,2008; Gustafsson et al., Trends Biotechnol 22:346-353, 2004.

In addition, other sequence modifications can be made to the nucleicacid sequences disclosed herein. For example, additional sequencemodifications are known to enhance gene expression in a cellular host.These include elimination of sequences encoding spurious polyadenylationsignals, exon/intron splice site signals, transposon-like repeats, andother such well-characterized sequences that may be deleterious to geneexpression. The G-C content of the sequence may also be adjusted tolevels average for a target cellular host, as calculated by reference toknown genes expressed in the host cell. In addition, the sequence can bemodified to avoid predicted hairpin secondary mRNA structures.

Other nucleic acid sequences may also be used in the preparation of theconstructs of the present disclosure, for example to enhance theexpression of the site directed nuclease coding sequence. Such nucleicacid sequences include the introns of the maize Adhl, intronl gene(Callis et al., Genes and Development 1:1183-1200, 1987), and leadersequences, (W-sequence) from the Tobacco Mosaic virus (TMV), MaizeChlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al., NucleicAcid Res. 15:8693-8711, 1987; and Skuzeski et al., Plant Mol. Biol.15:65-79, 1990). The first intron from the shrunken-1 locus of maize hasbeen shown to increase expression of genes in chimeric gene constructs.U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specificintrons in gene expression constructs, and Gallie et al. (Plant Physiol.106:929-939, 1994) also have shown that introns are useful forregulating gene expression on a tissue specific basis. To furtherenhance or to optimize site directed nuclease gene expression, the plantexpression vectors disclosed herein may also contain DNA sequencescontaining matrix attachment regions (MARs). Plant cells transformedwith such modified expression systems, then, may exhibit overexpressionor constitutive expression of a nucleotide sequence of the disclosure.

The expression constructs disclosed herein can also include nucleic acidsequences capable of directing the expression of the site directednuclease sequence to the chloroplast or other organelles and structuresin both prokaryotes and eukaryotes. Such nucleic acid sequences includechloroplast targeting sequences that encodes a chloroplast transitpeptide to direct the gene product of interest to plant cellchloroplasts. Such transit peptides are known in the art. With respectto chloroplast-targeting sequences, “operably linked” means that thenucleic acid sequence encoding a transit peptide (i.e., thechloroplast-targeting sequence) is linked to the site directed nucleasenucleic acid molecules disclosed herein such that the two sequences arecontiguous and in the same reading frame. See, for example, Von Heijneet al., Plant Mol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol.Chem. 264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol.84:965-968, 1987; Romer et al., Biochem. Biophys. Res. Commun.196:1414-1421, 1993; and Shah et al., Science 233:478-481, 1986.

Chloroplast targeting sequences are known in the art and include thechloroplast small subunit of ribulose-1,5-bisphosphate carboxylase(Rubisco) (de Castro Silva Filho et al., Plant Mol. Biol. 30:769-780,1996; Schnell et al., J. Biol. Chem. 266(5):3335-3342, 1991);5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al., J.Bioenerg. Biomemb. 22(6):789-810, 1990); tryptophan synthase (Zhao etal., J. Biol. Chem. 270(1 1):6081-6087, 1995); plastocyanin (Lawrence etal., J. Biol. Chem. 272(33):20357-20363, 1997); chorismate synthase(Schmidt et al., J. Biol. Chem. 268(36):27447-27457, 1993); and thelight harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.,J. Biol. Chem. 263:14996-14999, 1988). See also Von Heijne et al., PlantMol. Biol. Rep. 9:104-126, 1991; Clark et al., J. Biol. Chem.264:17544-17550, 1989; Della-Cioppa et al., Plant Physiol. 84:965-968,1987; Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421, 1993;and Shah et al., Science 233: 478-481, 1986.

In conjunction with any of the aspects, embodiments, methods and/orcompositions disclosed herein, the nucleic acid constructs may beprepared to direct the expression of the mutant site directed nucleasecoding sequence from the plant cell chloroplast. Methods fortransformation of chloroplasts are known in the art. See, for example,Svab et al., Proc. Nat'l. Acad. Sci. USA 87:8526-8530, 1990; Svab andMaliga, Proc. Nat'l. Acad. Sci. USA 90:913-917, 1993; Svab and Maliga,EMBO J. 12:601-606, 1993. The method relies on particle gun delivery ofDNA containing a selectable marker and targeting of the DNA to theplastid genome through homologous recombination. Additionally, plastidtransformation can be accomplished by transactivation of a silentplastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. Proc. Nat'l. Acad. Sci. USA91:7301-7305, 1994.

The nucleic acids of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

The nucleic acid constructs can be used to transform plant cells andregenerate transgenic plants comprising the site directed nucleasecoding sequences. Numerous plant transformation vectors and methods fortransforming plants are available. See, for example, U.S. Patent No.6,753,458, An, G. et al., Plant Physiol., 81:301-305, 1986; Fry, J. etal., Plant Cell Rep. 6:321-325, 1987; Block, M., Theor. Appl Genet.76:767-774, 1988; Hinchee et al., Stadler. Genet. Symp.203212.203-212,1990; Cousins et al., Aust. J. Plant Physiol. 18:481-494, 1991; Chee, P.P. and Slightom, J. L., Gene.118:255-260, 1992; Christou et al., Trends.Biotechnol. 10:239-246, 1992; D′Halluin et al., Bio/Technol. 10:309-314, 1992; Dhir et al., Plant Physiol. 99:81-88, 1992; Casas et al.,Proc. Nat'l. Acad Sci. USA 90:11212-11216, 1993; Christou, P., In VitroCell. Dev. Biol.-Plant 29P:1 19-124, 1993; Davies, et al., Plant CellRep. 12:180-183, 1993; Dong, J. A. and Mc Hughen, A., Plant Sci.91:139-148, 1993; Franklin, C. I. and Trieu, T. N., Plant. Physiol.102:167, 1993; Golovkin et al., Plant Sci. 90:41-52, 1993; Guo Chin Sci.Bull. 38:2072-2078; Asano, et al., Plant Cell Rep. 13, 1994; Ayeres N.M. and Park, W. D., Crit. Rev. Plant. Sci. 13:219-239, 1994; Barcelo etal., Plant. J. 5:583-592, 1994; Becker, et al., Plant. J. 5:299-307,1994; Borkowska et al., Acta. Physiol Plant. 16:225-230, 1994; Christou,P., Agro. Food. Ind. Hi Tech. 5:17-27, 1994; Eapen et al., Plant CellRep. 13:582-586, 1994; Hartman et al., Bio-Technology 12:919923, 1994;Ritala et al., Plant. Mol. Biol. 24:317-325, 1994; and Wan, Y. C. andLemaux, P. G., Plant Physiol. 104:3748, 1994. The constructs may also betransformed into plant cells using homologous recombination.

The term “wild-type” when made in reference to a peptide sequence andnucleotide sequence refers to a peptide sequence and nucleotide sequence(locus/gene/allele), respectively, which has the characteristics of thatpeptide sequence and nucleotide sequence when isolated from a naturallyoccurring source. A wild-type peptide sequence and nucleotide sequenceis that which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the peptidesequence and nucleotide sequence, respectively. “Wild-type” may alsorefer to the sequence at a specific nucleotide position or positions, orthe sequence at a particular codon position or positions, or thesequence at a particular amino acid position or positions.

“Consensus sequence” is defined as a sequence of amino acids ornucleotides that contain identical amino acids or nucleotides orfunctionally equivalent amino acids or nucleotides for at least 25% ofthe sequence. The identical or functionally equivalent amino acids ornucleotides need not be contiguous.

The term “Brassica” as used herein refers to plants of the Brassicagenus. Exemplary Brassica species include, but are not limited to, B.carinata, B. elongate, B. fruticulosa, B. juncea, B. napus, B. narinosa,B. nigra, B. oleracea, B. perviridis, B. rapa (syn B. campestris), B.rupestris, B. septiceps, and B. tournefortii.

A nucleobase is a base, which in certain preferred embodiments is apurine, pyrimidine, or a derivative or analog thereof. Nucleosides arenucleobases that contain a pentosefuranosyl moiety, e.g., an optionallysubstituted riboside or 2′-deoxyriboside. Nucleosides can be linked byone of several linkage moieties, which may or may not containphosphorus. Nucleosides that are linked by unsubstituted phosphodiesterlinkages are termed nucleotides. The term “nucleobase” as used hereinincludes peptide nucleobases, the subunits of peptide nucleic acids, andmorpholine nucleobases as well as nucleosides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; in someembodiments at least a portion of which can hybridize by Watson-Crickbase pairing to a DNA having the complementary sequence. Anoligonucleobase chain may have a single 5′ and 3′ terminus, which arethe ultimate nucleobases of the polymer. A particular oligonucleobasechain can contain nucleobases of all types. An oligonucleobase compoundis a compound comprising one or more oligonucleobase chains that may becomplementary and hybridized by Watson-Crick base pairing. Ribo-typenucleobases include pentosefuranosyl containing nucleobases wherein the2′ carbon is a methylene substituted with a hydroxyl, alkyloxy orhalogen. Deoxyribo-type nucleobases are nucleobases other than ribo-typenucleobases and include all nucleobases that do not contain apentosefuranosyl moiety.

In certain embodiments, an oligonucleobase strand may include botholigonucleobase chains and segments or regions of oligonucleobasechains. An oligonucleobase strand may have a 3′ end and a 5′ end, andwhen an oligonucleobase strand is coextensive with a chain, the 3′ and5′ ends of the strand are also 3′ and 5′ termini of the chain.

As used herein the term “codon” refers to a sequence of three adjacentnucleotides (either RNA or DNA) constituting the genetic code thatdetermines the insertion of a specific amino acid in a polypeptide chainduring protein synthesis or the signal to stop protein synthesis. Theterm “codon” is also used to refer to the corresponding (andcomplementary) sequences of three nucleotides in the messenger RNA intowhich the original DNA is transcribed.

As used herein, the term “homology” refers to sequence similarity amongproteins and DNA. The term “homology” or “homologous” refers to a degreeof identity. There may be partial homology or complete homology. Apartially homologous sequence is one that has less than 100% sequenceidentity when compared to another sequence.

“Heterozygous” refers to having different alleles at one or more geneticloci in homologous chromosome segments. As used herein “heterozygous”may also refer to a sample, a cell, a cell population or an organism inwhich different alleles at one or more genetic loci may be detected.Heterozygous samples may also be determined via methods known in the artsuch as, for example, nucleic acid sequencing. For example, if asequencing electropherogram shows two peaks at a single locus and bothpeaks are roughly the same size, the sample may be characterized asheterozygous. Or, if one peak is smaller than another, but is at leastabout 25% the size of the larger peak, the sample may be characterizedas heterozygous. In some embodiments, the smaller peak is at least about15% of the larger peak. in other embodiments, the smaller peak is atleast about 10% of the larger peak. In other embodiments, the smallerpeak is at least about 5% of the larger peak. In other embodiments, aminima amount of the smaller peak is detected.

As used herein, “homozygous” refers to having identical alleles at oneor more genetic loci in homologous chromosome segments. “Homozygous” mayalso refer to a sample, a cell, a cell population or an organism inwhich the same alleles at one or more genetic loci may be detected.Homozygous samples may be determined via methods known in the art, suchas, for example, nucleic acid sequencing. For example, if a sequencingelectropherograms shows a single peak at a particular locus, the samplemay be termed “homozygous” with respect to that locus.

The term “hemizygous” refers to a gene or gene segment being presentonly once in the genotype of a cell or an organism because the secondallele is deleted, or is not present on the homologous chromosomesegment. As used herein “hemizygous” may also refer to a sample, a cell,a cell population or an organism in which an allele at one or moregenetic loci may be detected only once in the genotype.

The term “zygosity status” as used herein refers to a sample, a cellpopulation, or an organism as appearing heterozygous, homozygous, orhemizygous as determined by testing methods known in the art anddescribed herein. The term “zygosity status of a nucleic acid” meansdetermining whether the source of nucleic acid appears heterozygous,homozygous, or hemizygous. The “zygosity status” may refer todifferences in at a single nucleotide position in a sequence. in somemethods, the zygosity status of a sample with respect to a singlemutation may be categorized as homozygous wild-type, heterozygous (i.e.,one wild-type allele and one mutant allele), homozygous mutant, orhemizygous a single copy of either the wild-type or mutant allele).

As used herein, the term “RTDS” refers to The Rapid. Trait DevelopmentSystem™ (RTDS) developed by Cibus. RTDS is a site-specific genemodification system that is effective at making precise changes in agene sequence without the incorporation of foreign genes or controlsequences.

The term “about” as used herein means in quantitative terms plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%. Moreover, where “about” is used herein inconjunction with a quantitative term it is understood that in additionto the value plus or minus 10%, the exact value of the quantitative termis also contemplated and described. For example, the term “about 3%”expressly contemplates, describes and includes exactly 3%.

RTDS and Repair Oligonucleotides (GRONs)

This disclosure generally relates to novel methods to improve theefficiency of the targeting of modifications to specific locations ingenomic or other nucleotide sequences. Additionally, this disclosurerelates to target DNA that has been modified, mutated or marked by theapproaches disclosed herein. The disclosure also relates to cells,tissue, and organisms which have been modified by the disclosure'smethods. The present disclosure builds on the development ofcompositions and methods related in part to the successful conversionsystem, the Rapid Trait Development System (RTDS™, Cibus US LLC).

RTDS is based on altering a targeted gene by utilizing the cell's owngene repair system to specifically modify the gene sequence in situ andnot insert foreign DNA and gene expression control sequences. Thisprocedure effects a precise change in the genetic sequence while therest of the genome is left unaltered. In contrast to conventionaltransgenic GMOs, there is no integration of foreign genetic material,nor is any foreign genetic material left in the plant. The changes inthe genetic sequence introduced by RTDS are not randomly inserted. Sinceaffected genes remain in their native location, no random, uncontrolledor adverse pattern of expression occurs.

The RTDS that effects this change is a chemically synthesizedoligonucleotide (GRON) as described herein which may be composed of bothDNA and modified RNA bases as well as other chemical moieties, and isdesigned to hybridize at the targeted gene location to create amismatched base,-pair(s). This mismatched base-pair acts as a signal toattract the cell's own natural gene repair system to that site andcorrect (replace, insert or delete) the designated nucleotide(s) withinthe gene. Once the correction process is complete the RTDS molecule isdegraded and the now-modified or repaired gene is expressed under thatgene's normal endogenous control mechanisms.

The methods and compositions disclosed herein can be practiced or madewith “gene repair oligonucleobases” (GRON) having the conformations andchemistries as described in detail herein and below. The “gene repairoligonucleohases” as contemplated herein have also been described inpublished scientific and patent literature using other names including“recombinagenic oligonucleohases;” “RNA/DNA chimeric oligonucleotides;”“chimeric oligonucleotides;” “mixed duplex oligonucleotides” (MDONs);“RNA DNA oligonucleotides (RDOs);” “gene targeting oligonucleotides;”“genoplasts;” “single stranded modified oligonucleotides;” “Singlestranded oligodeoxynucleotide, mutational vectors” (SSOMVs); “duplexmutational vectors;” and “heteroduplex mutational vectors.” The generepair oligonucleobase can be introduced into a plant cell using anymethod commonly used in the art, including but not limited to,microcarriers (biolistic delivery), microfibers, polyethylene glycol(PEG)-mediated uptake, electroporation, and microinjection.

In one embodiment, the gene repair oligonucleobase is a mixed duplexoligonucleotides (MDON) in which the RNA-type nucleotides of the mixedduplex oligonucleotide are made RNase resistant by replacing the2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing asubstituent on the 2′-O. Suitable substituents include the substituentstaught by the Kmiec II. Alternative substituents include thesubstituents taught by U.S. Pat. No. 5,334,711 (Sproat) and thesubstituents taught by patent publications EP 629 387 and EP 679 657(collectively, the Martin Applications), which are hereby incoii'oratedby reference. As used herein, a 2′-fluoro, chloro or bromo derivative ofa ribonucleotide or a ribonucleotide having a T-OH substituted with asubstituent described in the Martin Applications or Sproat is termed a“T-Substituted Ribonucleotide.” As used herein the term “RNA-typenucleotide” means a T-hydroxyl or 2′-Substituted Nucleotide that islinked to other nucleotides of a mixed duplex oligonucleotide by anunsubstituted phosphodiester linkage or any of the non-natural linkagestaught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-typenucleotide” means a nucleotide having a T-H, which can be linked toother nucleotides of a gene repair oligonucleobase by an unsubstitutedphosphodiester linkage or any of the non-natural linkages taught byKmiec I or Kmiec II.

In a particular embodiment of the present disclosure, the gene repairoligonucleobase is a mixed duplex oligonucleotide (MDON) that is linkedsolely by unsubstituted phosphodiester bonds. In alternativeembodiments, the linkage is by substituted phosphodiesters,phosphodiester derivatives and non-phosphorus-based linkages as taughtby Kmiec II. In yet another embodiment, each RNA-type nucleotide in themixed duplex oligonucleotide is a 2′-Substituted Nucleotide. Particularpreferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro,T-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy,2′-methoxyethyloxy, T-fluoropropyloxy and 2′-trifluoropropyloxysubstituted ribonucleotides. More preferred embodiments of2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy,2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In anotherembodiment the mixed duplex oligonucleotide is linked by unsubstitutedphosphodiester bonds,

Although mixed duplex oligonucleotides (MDONs) having only a single typeof 2′-substituted RNA-type nucleotide are more conveniently synthesized,the methods of the disclosure can be practiced with mixed duplexoligonucleotides having two or more types of RNA-type nucleotides. Thefunction of an RNA segment may not be affected by an interruption causedby the introduction of a deoxynucleotide between two RNA-typetrinucleotides, accordingly, the term RNA segment encompasses terms suchas “interrupted RNA segment.” An uninterrupted RNA segment is termed acontiguous RNA segment. In an alternative embodiment an RNA segment cancontain alternating RNase-resistant and unsubstituted 2′-OH nucleotides.The mixed duplex oligonucleotides in some embodiments have fewer than100 nucleotides and other embodiments fewer than 85 nucleotides, butmore than 50 nucleotides. The first and second strands are Watson-Crickbase paired. In one embodiment the strands of the mixed duplexoligonucleotide are covalently bonded by a linker, such as a singlestranded hexa, penta or tetranucleotide so that the first and secondstrands are segments of a single oligonucleotide chain having a single3′ and a single 5′ end. The 3′ and 5′ ends can be protected by theaddition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotidesare Watson-Crick paired to adjacent nucleotides. A second hairpin capcan, additionally, be placed at the junction between the first andsecond strands distant from the 3′ and 5′ ends, so that the Watson-Crickpairing between the first and second strands is stabilized.

The first and second strands contain two regions that are homologouswith two fragments of the target gene/allele, i.e., have the samesequence as the target gene/allele. A homologous region contains thenucleotides of an RNA segment and may contain one or more DNA-typenucleotides of connecting DNA segment and may also contain DNA-typenucleotides that are not within the intervening DNA segment. The tworegions of homology are separated by, and each is adjacent to, a regionhaving a sequence that differs from the sequence of the target gene,termed a “heterologous region.” The heterologous region can contain one,two or three mismatched nucleotides. The mismatched nucleotides can hecontiguous or alternatively can be separated by one or two nucleotidesthat are homologous with the target gene/allele. Alternatively, theheterologous region can also contain an insertion or one, two, three orof five or fewer nucleotides. Alternatively, the sequence of the mixedduplex oligonucleotide may differ from the sequence of the targetgene/allele only by the deletion of one, two, three, or five or fewernucleotides from the mixed duplex oligonucleotide. The length andposition of the heterologous region is, in this case, deemed to be thelength of the deletion, even though no nucleotides of the mixed duplexoligonucleotide are within the heterologous region. The distance betweenthe fragments of the target gene that are complementary to the twohomologous regions is identical to the length of the heterologous regionwhere a substitution or substitutions is intended. When the heterologousregion contains an insertion, the homologous regions are therebyseparated in the mixed duplex oligonucleotide farther than theircomplementary homologous fragments are in the gene/allele, and theconverse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part ofa homologous region, i.e., a region that is identical in sequence to afragment of the target gene, which segments together in some embodimentscontain at least 13 RNA-type nucleotides and in some embodiments from 16to 25 RNA-type nucleotides or yet other embodiments 18-22 RNA-typenucleotides or in some embodiments 20 nucleotides. In one embodiment,RNA segments of the homology regions are separated by and adjacent to,i.e., “connected by” an intervening DNA segment. In one embodiment, eachnucleotide of the heterologous region is a nucleotide of the interveningDNA segment. An intervening DNA segment that contains the heterologousregion of a mixed duplex oligonucleotide is termed a “mutator segment.”

In another embodiment of the present disclosure, the gene repairoligonucleobase (GRON) is a single stranded oligodeoxynucleotidemutational vector (SSOMV), such as disclosed in International PatentApplication PCT/USOO/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and7,060,500 which is incorporated by reference in its entirety. Thesequence of the SSOMV is based on the same principles as the mutationalvectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012;5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 andin International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723;WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains tworegions that are homologous with the target sequence separated by aregion that contains the desired genetic alteration termed the imitatorregion. The Imitator region can have a sequence that is the same lengthas the sequence that separates the homologous regions in the targetsequence, but having a different sequence. Such a mutator region cancause a substitution. Alternatively, the homologous regions in the SSOMVcan be contiguous to each other, while the regions in the target genehaving the same sequence are separated by one. two or more nucleotides.Such an SSOMV causes a deletion from the target gene of the nucleotidesthat are absent from the SSOMV. Lastly, the sequence of the target genethat is identical to the homologous regions may be adjacent in thetarget gene but separated by one, two, or more nucleotides in thesequence of the SSOMV. Such an SSOMV causes an insertion in the sequenceof the target gene. In certain embodiments, a SSOMV does not anneal toitself.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked byunmodified phosphodiester bonds except that the 3′ terminal and/or 5′terminal internucleotide linkage or alternatively the two 3′ terminaland/or 5′ terminal internucleotide linkages can be a phosphorothioate orphosphoamidate. As used herein an internucleotide linkage is the linkagebetween nucleotides of the SSOMV and does not include the linkagebetween the 3′ end nucleotide or 5′ end nucleotide and a blockingsubstituent. In a specific embodiment the length of the SSOMV is between21 and 55 deoxynucleotides and the lengths of the homology regions are,accordingly, a total length of at least 20 deoxynucleotides and at leasttwo homology regions should each have lengths of at least 8deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding orthe non-coding strand of the target gene. When the desired mutation is asubstitution of a single base, it is preferred that both the mutatornucleotide and the targeted nucleotide be a pyrimidine. To the extentthat is consistent with achieving the desired functional result, it ispreferred that both the mutator nucleotide and the targeted nucleotidein the complementary strand be pyrimidines. Particularly preferred areSSOMVs that encode transversion mutations, i.e., a C or T mutatornucleotide is mismatched, respectively, with a C or T nucleotide in thecomplementary strand.

Okazaki Fragment/2′-OME GRON Design. In various embodiments, a GRON mayhave both RNA and DNA nucleotides and/or other types of nucleobases. Insome embodiments, one or more of the DNA or RNA nucleotides comprise amodification. In certain embodiments, the first 5′ nucleotide is an RNAnucleotide and the remainder of the nucleotides are DNA. In stillfurther embodiments, the first 5′ RNA nucleotide is modified with a2-O-Me. In other embodiments, the first two, three, four, five, six,seven, eight, nine, ten or more 5′ nucleotides are an RNA nucleotide andthe remainder of the nucleotides are DNA. In still further embodiments,one or more of the first two, three, four, five, six, seven, eight,nine, ten or more 5′ RNA nucleotide are modified with a 2-O-Me. In plantcells, double-strand beaks in DNA are typically repaired by the NHEJ DNArepair pathway. This pathway does not require a template to repair theDNA and is therefore error prone. The advantage of using this pathway torepair DNA for a plant cell is that it is quick, ubiquitous and mostimportantly can occur at times when a cell is not undergoing DNAreplication. Another DNA repair pathway that functions in repairingdouble-strand breaks outside of the replication fork in plant cells iscalled homologous recombination (HR); however, unlike the NHEJ pathwaythis type of repair is precise and requires the use of a DNA template(GRON). Since these GRONs mimic Okazaki fragments at the DNA replicationfork of targeted genes, it is not obvious to use them with adouble-strand DNA cutter to those skilled in the art.

Improving Efficiency

The present disclosure provides a number of approaches to increase theeffectiveness of conversion of a target gene using repairoligonucleotides, and which may be used alone or in combination with oneanother. These include:

-   -   1. Introducing modifications to the repair oligonucleotides        which attract DNA repair machinery to the targeted (mismatch)        site.        -   A. Introduction of one or more abasic sites in the            oligonucleotide (e.g., within 10 bases, and in some            embodiments with 5 bases of the desired mismatch site)            generates a lesion which is an intermediate in base excision            repair (BER), and which attracts BER machinery to the            vicinity of the site targeted for conversion by the repair            oligonucleotide. dSpacer (abasic furan) modified            oligonucleotides may be prepared as described in, for            example, Takeshita et al., J. Biol. Chem., 262:10171-79,            1987.        -   B. Inclusion of compounds which induce single or double            strand breaks, either into the oligonucleotide or together            with the oligonucleotide, generates a lesion which is            repaired by NHEJ, microhomology-mediated end joining (MMEJ),            and homologous recombination. By way of example, the            bleomycin family of antibiotics, zinc fingers, FokI (or any            type IIS class of restriction enzyme) and other nucleases            may be covalently coupled to the 3′ or 5′ end of repair            oligonucleotides, in order to introduce double strand breaks            in the vicinity of the site targeted for conversion by the            repair oligonucleotide. The bleomycin family of antibiotics            are DNA cleaving glycopeptides which include bleomycin,            zeocin, phleomycin, tallysomycin, pepleomycin and others.        -   C. Introduction of one or more 8′oxo dA or dG incorporated            in the oligonucleotide (e.g., within 10 bases, and in some            embodiments with 5 bases of the desired mismatch site)            generates a lesion which is similar to lesions created by            reactive oxygen species. These lesions induce the so-called            “pushing repair” system. See, e.g., Kim et al., J. Biochem.            Mol. Biol. 37:657-62, 2004.    -   2. Increase stability of the repair oligonucleotides:        -   Introduction of a reverse base (idC) at the 3′ end of the            oligonucleotide to create a 3′ blocked end on the repair            oligonucleotide.        -   Introduction of one or more 2′O-methyl nucleotides or bases            which increase hybridization energy (see, e.g.,            WO2007/073149) at the 5′ and/or 3′ of the repair            oligonucleotide.        -   Introduction of one or a plurality of 2′O-methyl RNA            nucleotides at the 5′ end of the repair oligonucleotide,            leading into DNA bases which provide the desired mismatch            site, thereby creating an Okazaki Fragment-like nucleic acid            structure.        -   Conjugated (5′ or 3′) intercalating dyes such as acridine,            psoralen, ethidium bromide and Syber stains.        -   Introduction of a 5′ terminus cap such as a T/A clamp, a            cholesterol moiety, SIMA (HEX), riboC and amidite.        -   Backbone modifications such as phosphothioate, 2′-O methyl,            methyl phosphonates, locked nucleic acid (LNA), MOE            (methoxyethyl), di PS and peptide nucleic acid (PNA).        -   Crosslinking of the repair oligonucleotide, e.g., with            intrastrand crosslinking reagents agents such as cisplatin            and mitomycin C.        -   Conjugation with fluorescent dyes such as Cy3, DY547, Cy3.5,            Cy3B, Cy5 and DY647.    -   3. Increase hybridization energy of the repair oligonucleotide        through incorporation of bases which increase hybridization        energy (see, e.g., WO2007/073149).    -   4. Increase the quality of repair oligonucleotide synthesis by        using nucleotide multimers (dimers, trimers, tetramers, etc.) as        building blocks for synthesis. This results in fewer coupling        steps and easier separation of the full length products from        building blocks.    -   5. Use of long repair oligonucleotides (i.e., greater than 55        nucleotides in length, for example such as the lengths described        herein, for example having one or more mutations or two or more        mutations targeted in the repair oligonucleotide.

Examples of the foregoing approaches are provided in Table 1.

TABLE 1 Exemplary GRON chemistries. Oligo type Modifications 5′ mods T/Aclamp T/A clamp Backbone modifications Phosphothioate PS Intercalatingdyes 5′ Acridine 3′ idC Acridine, idC 2′-O-methyl DNA/RNA Cy3replacements DY547 Facilitators 2′-O-Me oligos designed 5′ 2′-O-Me and3′ of the converting oligo Abasic Abasic site placed in various Abasic 2locations 5′ and 3′ to the converting base. 44 mer Assist Assistapproach Cy3, idC on one, none on the Overlap: other: 2 oligos: 1 withCy3/idC, 1 unmodified repair oligo Assist Assist approach only make theunmodified No overlap: oligo 2 oligos: 1 with Cy3/idC, 1 unmodifiedrepair oligo Abasic THF site placed in various Tetrahydrofuran (dspacer) locations 5′ and 3′ to the converting base. 44 mer Backbonemodifications 9 2′-O-Me Trimers Trimer amidites, Cy3, idC Pushing repair8′oxo dA, 5′ Cy3, idC Pushing repair 8′oxo dA, 5′ Cy3, idC Double StrandBreak Bleomycin Crosslinker Cisplatin Crosslinker Mitomycin CFacilitators super bases 5′ and 3′ of 2 amino dA and 2-thio T convertingoligo Super oligos 2′amino d, 5′ Cy3, idC Super oligos 2-thio T, 5′ Cy3,idC Super oligos 7-deaza A, 5′ Cy3, idC Super oligos 7-deaza G, 5′ Cy3,idC Super oligos propanyl dC, 5′ Cy3, idC Intercalating dyes 5′Psoralen/3′ idC Psoralen, idC Intercalating dyes 5′ Ethidium bromideEthidium bromide Intercalating dyes 5′ Syber stains Syber stains 5′ mods5′ Chol/3′ idC Cholesterol Double mutation Long oligo (55′ bases) w/ Anymodification 2 mutation 5′ mods 5′ SIMA HEX/3′idC SIM HEX, idC Backbonemodifications 9 Methyl phosphonates Backbone modifications LNA Backbonmodifications 1 MOE (methoxyethyl) Cy3 replacements Cy35 Cy3replacements Cy5 Backbone modifications di PS 5′ mods riboC for branchnm Backbone modifications PNA Cy3 replacements DY647 5′ mods 5′ branchsymmetric branch amidite/idC

The foregoing modifications may also include known nucleotidemodifications such as methylation, 5′ intercalating dyes, modificationsto the 5′ and 3′ ends, backbone modifications, crosslinkers, cyclizationand ‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analog such as inosine. Modifications of nucleotidesinclude the addition of acridine, amine, biotin, cascade blue,cholesterol, Cy3 @, Cy5 @, Cy5.5 @ Daboyl, digoxigenin, dinitrophenyl,Edans, 6-FAM, fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE,phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET,AMCA-S″, SE, BODIPY°, Marina Blue@, Pacific Blue@, Oregon Green@,Rhodamine Green@, Rhodamine Red@, Rhodol Green@ and Texas Red@.Polynucleotide backbone modifications include methylphosphonate,2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Basemodifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N6-Me-dA, abasic site(dSpacer), biotin dT, 2′-OMe-SMe-C, 2′-OMe-propynyl-C, 3′-(5-Me-dC),3′-(ddC), 5-Br-dC, 5-1-duc, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA,convertible dC, convertible dG, convertible dT, convertible dU,7-deaza-dG, 8-Br-dG, 8-oxo-dG, O6-Me-dG, S6-DNP-dG, 4-methyl-indole,5-nitroindole, 2′-OMe-inosine, 2′-dl, o6-phenyl-dl, 4-methyl-indole,2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP (purine analogue),dK (pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT,biotin-dT, carboxy-dT, 04-Me-dT, 04-triazol dT, 2′-OMe-propynyl-U,5-Br-dU, 2′-dU, 5-F-dU, 5-1-dU, 04-triazol dU. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than asugar. PNAs mimic the behavior of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful biomolecular tools, antisense and antigene agents,molecular probes and biosensors.

Oligonucleobases may have nick(s), gap(s), modified nucleotides such asmodified oligonucleotide backbones, abasic nucleotides, or otherchemical moieties. In a further embodiment, at least one strand of theoligonucleobase includes at least one additional modified nucleotide,e.g., a 2′-O-methyl modified nucleotide such as a MOE (methoxyethyl), anucleotide having a 5′-phosphorothioate group, a terminal nucleotidelinked to a cholesteryl derivative, a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide (the nucleobase is missing or has a hydroxyl group inplace thereof (see, e.g., Glen Research,http://www.glenresearch.com/GlenReports/GR21-14.html)), a2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidite, and a non-natural basecomprising nucleotide. Various salts, mixed salts and free acid formsare also included.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphoro-dithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3 ‘-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkyl-phosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3’-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the 3‘-most internucleotide linkage i.e. a single inverted nucleoside residuewhich may be abasic (the nucleobase is missing or has a hydroxyl groupin place thereof). The most common use of a linkage inversion is to adda 3’-3′ linkage to the end of an antisense oligonucleotide with aphosphorothioate backbone. The 3′-3′ linkage further stabilizes theantisense oligonucleotide to exonuclease degradation by creating anoligonucleotide with two 5′-OH ends and no 3′-OH end. Linkage inversionscan be introduced into specific locations during oligonucleotidesynthesis through use of “reversed phosphoramidites”. These reagentshave the phosphoramidite groups on the 5′-OH position and thedimethoxytrityl (DMT) protecting group on the 3′-OH position. Normally,the DMT protecting group is on the 5′-OH and the phosphoramidite is onthe 3′-OH.

Examples of modified bases include, but are not limited to,2-aminopurine, 2′-amino-butyryl pyrene-uridine, 2 ′-aminouridine, 2′-deoxyuridine, 2′-fluoro-cytidine, 2 ′-fluoro-uridine,2,6-diaminopurine, 4-thio-uridine, 5-bromo-uridine, 5-fluoro-cytidine,5-fluorouridine, 5-indo-uridine, 5-methyl-cytidine, inosine,N3-methyl-uridine, 7-deaza-guanine, 8-aminohexyl-amino-adenine,6-thio-guanine, 4-thio-thymine, 2-thio-thymine, 5-iodo-uridine,5-iodo-cytidine, 8-bromo-guanine, 8-bromo-adenine, 7-deaza-adenine,7-diaza-guanine, 8-oxo-guanine, 5,6-dihydro-uridine, and5-hydroxymethyl-uridine. These synthetic units are commerciallyavailable; (for example, purchased from Glen Research Company) and canbe incorporated into DNA by chemical synthesis.

Examples of modification of the sugar moiety are 3′-deoxylation,2′-fluorination, and arabanosidation, however, it is not to be construedas being limited thereto. Incorporation of these into DNA is alsopossible by chemical synthesis.

Examples of the 5′ end modification are 5′-amination, 5′-biotinylation,5′-fluoresceinylation, 5′-tetrafluoro-fluoreceinyaltion, 5′-thionation,and 5′-dabsylation, however it is not to be construed as being limitedthereto.

Examples of the 3′ end modification are 3′-amination, 3′-biotinylation,2,3-dideoxidation, 3 ′-thionation, 3 ′-dabsylation, 3 ′-carboxylation,and 3 ′-cholesterylation, however, it is not to be construed as beinglimited thereto.

In one preferred embodiment, the oligonucleobase can contain a 5′blocking substituent that is attached to the 5′ terminal carbons througha linker. The chemistry of the linker is not critical other than itslength, which should in some embodiments be at least 6 atoms long andthat the linker should be flexible. A variety of non-toxic substituentssuch as biotin, cholesterol or other steroids or a non-intercalatingcationic fluorescent dye can be used. Particularly preferred reagents tomake oligonucleobases are the reagents sold as Cy3™ and Cy5™ by GlenResearch, Sterling Va. (now GE Healthcare), which are blockedphosphoroamidites that upon incorporation into an oligonucleotide yield3,3,3′,3′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanineand indodicarbocyanine dyes, respectively. Cy3 is particularlypreferred. When the indocarbocyanine is N-oxyalkyl substituted it can beconveniently linked to the 5′ terminal of the oligodeoxynucleotide as aphosphodiester with a 5′ terminal phosphate. When the commerciallyavailable Cy3 phosphoramidite is used as directed, the resulting 5′modification consists of a blocking substituent and linker togetherwhich are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethylindomonocarbocyanine. Other dyes contemplated include Rhodamine6G,Tetramethylrhodamine, Sulforhodamine 101 Merocyanine 540, Atto565,Atto550 26, Cy3.5, Dy547 Dy54$, Dy549, Dy554, Dy555, Dy556, Dy560,mStrawberry and mCherry.

In a preferred embodiment the indocarhocyanine dye is tetra substitutedat the 3 and 3′ positions of the indole rings. Without limitations as totheory these substitutions prevent the dye from being an intercalatingdye. The identity of the substituents at these positions is notcritical.

The oligo designs herein described might also be used as more efficientdonor templates an combination with other DNA editing or recombinationtechnologies including, but not limited to, gene targeting usingsite-specific homologous recombination by zinc finger nucleases,Transcription Activator-Like Effector Nucleases (TALENs) or ClusteredRegularly interspaced Short Palindromic Repeats (CRISPRs).

The present disclosure in certain aspects and embodiments generallyrelates to methods for the efficient modification of genomic cellularDNA and/or recombination of DNA into the genomic DNA of cells. Althoughnot limited to any particular use, some methods provided herein may incertain embodiments he useful in, for example, introducing amodification into the genome of a cell for the purpose of determiningthe effect of the modification on the cell. For example, a modificationmay be introduced into the nucleotide sequence which encodes an enzymeto determine whether the modification alters the enzymatic activity ofthe enzyme, and/or determine the location of the enzyme's catalyticregion. Alternatively, the modification may he introduced into thecoding sequence of a DNA-binding protein to determine whether the DNAbinding activity of the protein is altered, and thus to delineate theparticular DNA-binding region within the protein. Yet anotheralternative is to introduce a modification into a non-coding regulatorysequence (e.g., promoter, enhancer, regulatory RNA sequence (miRNA),etc.) in order to determine the effect of the modification on the levelof expression of a second sequence which is operably linked to thenon-coding regulatory sequence. This may be desirable to, for example,define the particular sequence which possesses regulatory activity.

DNA Cutters

One strategy for producing targeted gene disruption is through thegeneration of single strand or double strand DNA breaks using a DNAcutter such as a site-specific endonuclease. Endonucleases are mostoften used for targeted gene disruption in organisms that havetraditionally been refractive to more conventional gene targetingmethods, such as algae, plants, and large animal models, includinghumans. For example, there are currently human clinical trials underwayinvolving zinc finger nucleases for the treatment and prevention of HIVinfection. Additionally, endonuclease engineering is currently beingused in attempts to disrupt genes that produce undesirable phenotypes incrops.

Zinc Fingers

One class of artificial endonucleases is the zinc finger endonucleases.Zinc finger endonucleases combine a non-specific cleavage domain,typically that of FokI endonuclease, with zinc finger protein domainsthat are engineered to bind to specific DNA sequences. The modularstructure of the zinc finger endonucleases makes them a versatileplatform for delivering site-specific double-strand breaks to thegenome. As FokI endonuclease cleaves as a dimer, one strategy to preventoff-target cleavage events has been to design zinc finger domains thatbind at adjacent 9 base pair sites. See also U.S. Pat. Nos. 7,285,416;7,521,241; 7,361,635; 7,273,923; 7,262,054; 7,220,719; 7,070,934;7,013,219; 6,979,539; 6,933,113; 6,824,978; each. of which is herebyherein incorporated by reference in its entirety.

TALENs

TALENs are targetable nucleases are used to induce single- anddouble-strand breaks into specific DNA sites, which are then repaired bymechanisms that can be exploited to create sequence alterations at thecleavage site.

The fundamental building block that is used to engineer the DNA-bindingregion of TALENs is a highly conserved repeat domain derived fromnaturally occurring TALEs encoded by Xanthomonas spp. proteobacteria.DNA binding by a TALEN is mediated by arrays of highly conserved 33-35amino acid repeats that are flanked by additional TALE-derived domainsat the amino-terminal and carboxy-terminal ends of the repeats.

These TALE repeats specifically bind to a single base of DNA, theidentity of which is determined by two hypervariable residues typicallyfound at positions 12 and 13 of the repeat, with the number of repeatsin an array corresponded to the length of the desired target nucleicacid, the identity of the repeat selected to match the target nucleicacid sequence. In some embodiments, the target nucleic acid is between15 and 20 base pairs in order to maximize selectivity of the targetsite. Cleavage of the target nucleic acid typically occurs within 50base pairs of TALEN binding. Computer programs for TALEN recognitionsite design have been described in the art. See, e.g., Cermak et al.,Nucleic Acids Res. 2011 July; 39(12): e82.

Once designed to match the desired target sequence, TALENs can beexpressed recombinantly and introduced into protoplasts as exogenousproteins, or expressed from a plasmid within the protoplast oradministered as mRNA.

Meganueleases

The homing endonucleases, also known as meganucleases, are sequencespecific endonucleases that generate double strand breaks in genomic DNAwith a high degree of specificity due to their large (e.g., >14 bp)cleavage sites. While the specificity of the homing endonucleases fortheir target sites allows for precise targeting of the induced DNAbreaks, homing endonuclease cleavage sites are rare and the probabilityof finding a naturally occurring cleavage site in a targeted gene islow.

Another class of artificial endonucleases is the engineeredmeganucleases. Engineered homing endonucleases are generated bymodifying the specificity of existing homing endonucleases. in oneapproach, variations are introduced in the amino acid sequence ofnaturally occurring homing endonucleases and then the resultantengineered horning endonucleases are screened to select functionalproteins which cleave a targeted binding site. in another approach,chimeric homing endonucleases are engineered by combining therecognition sites of two different homing endonucleases to create a newrecognition site composed of a half-site of each homing endonuclease.See e.g., U.S. Pat. No. 8,338,157.

CRISPRs or CRISPR/Cas Systems

CRISPR-Cas system contains three basic design components: 1) Cas gene,transcript (e.g., mRNA) or protein; 2) guide RNA (gRNA); and 3) crRNAs(CRISPR RNA) are RNA segments processed from RNA transcripts encodingthe CRISPR repeat arrays, which harbor a “protospacer” region that arecomplementary to a foreign DNA site (e.g., endogenous DNA target region)and a part of the CRISPR repeat. See e.g., PCT Application Nos.WO/2014/093661 and WO/2013/176772.

Cas (CRISPR Associated) Gene, Transcript (e.g., mRNA) or Protein

Transient Cas expression from a plasmid vector, direct delivery of Casprotein and or direct delivery of Cas mRNA into plant cells. Cas genesare codon optimized for expression in higher plants, algae or yeast andare driven by either a constitutive, inducible, tissue-specific orspecies-specific promoter when applicable. Cas transcript terminationand polyadenlyation signals are either NosT, RBCT, HSP18.2T or othergene specific or species-specific terminators. Cas gene cassettes ormRNA may contain introns, either native or in combination withgene-specific promoters and or synthetic promoters. Cas protein maycontain one or more nuclear localization signal sequences (NLS),mutations, deletions, alterations or truncations. In transientexpression systems, Cas gene cassettes may be combined with othercomponents of the CRISPR-Cas system such as gRNA cassettes on the sametransient expression vector. Alternatively, Cas gene cassettes may belocated and expressed from constructs independent of gRNA cassettes orfrom other components of the CRISPR-Cas system. CRISPR associated (Cas)gene-encode for proteins with a variety of predicted nucleicacid-manipulating activities such as nucleases, helicases andpolymerase. Cas genes include cas9. Cas9 is a gene encoding a largeprotein containing a predicted RuvC-like and HNH endonuclease domainsand is associated with the CRISPR adaptive immunity system that ispresent in most archaea and many bacteria. Cas9 protein consists of twolobes:

-   -   1) Recognition (REC) lobe—consists of three domains:        -   a) BH (bridge helix)        -   b) REC1—facilitates RNA-guided DNA targeting        -   c) REC2—facilitates RNA-guided DNA targeting    -   2) Nuclease (NUC) lobe—consists of three domains:        -   a) RuvC—facilitates RNA-guided DNA targeting; endonuclease            activity        -   b) HNH—endonuclease activity        -   c) PI—PAM interacting

In other embodiments, the cas gene may be a homolog of cas9 in which theRuvC, HNH, REC and BH domains are highly conserved. In some embodiments,cas genes are those from the following species.

Guide RNA (gRNA)

gRNA or sgRNA (single guide RNA) is engineered as a fusion between acrRNA and part of the transactivating CRISPR RNA (tracrRNA) sequence,which guides the Cas9 to a specific target DNA sequence that iscomplementary to the protospacer region. Guide RNA may include anexpression cassette containing a chimeric RNA design with a longtracerRNA hybrid, short tracrRNA hybrid or a native CRISPRarray+tracrRNA conformation. Chimeric gRNA combines the targetingspecificity of the crRNA with the scaffolding properties of the tracrRNAinto a single transcript. gRNA transient expression is controlled byspecies-specific higher plant RNA Polymerase III promoters such as thosefrom the U6 or U3 snRNA gene family (Wang et al 2008). gRNA transcripttermination is controlled by a 6-20 nucleotide tract of poly dT as perWang et al 2008. gRNA expression cassettes are located on the same ordifferent transient expression vectors from other components of theCRISPR-Cas system. gRNA transcripts may be synthesized in-vitro anddelivered directly into plant cells, independent of or in combinationwith gRNA transient expression vectors.

Target region

Guide RNAs contain two components that define specificity to a DNAtarget region, a proto-spacer and a proto-spacer adjacent motif (PAM).Proto-spacer sequence, typically 20 nucleotides but can vary based onthe DNA target, provides DNA sequence specificity for the CRISPR-Cascomplex. DNA targets also contain a NNG or NAG tri-nucleotide sequence(PAM) where N denotes any nucleotide, immediately 3′ or downstream ofthe proto-spacer.

One Component Approach

Similar to Le Cong et al. (2013) and others, a simplified “one componentapproach” to CRISPR-Cas gene editing wherein a single transientexpression construct contains all components of the CRISPR-Cas complex,i.e. both the gRNA and the Cas expressions cassettes. This allows for aneasy modular design for targeting single or multiple loci in any givenplant or crop. Targeting multiple loci can be achieved by simplyswapping in the target-specific gRNA cassettes. Additionally, speciesspecific promoters, terminators or other expressing enhancing elementscan easily be shuttled in and out of “one component approach” transientvectors allowing for optimal expression of both gRNA and Cas protein ina species specific manner.

Two Component Approach

In the two component approach, Cas and gRNA expression cassettes arelocated on different transient expression vectors. This allows fordelivery of a CRISPR-Cas editing components separately, allowing fordifferent ratios of gRNA to Cas within the same cell. Similar to the onecomponent approach, the two component approach also allows forpromoters, terminators or other elements affecting expression ofCRISPR-Cas components to be easily altered and allow targeting of DNA ina species-specific manner.

Antibiotics

Another class of endonucleases are antibiotics which are DNA cleavingglycopeptides such as the bleomycin family of antibiotics are DNAcleaving glycopeptides which include bleomycin, zeocin, phleomycin,tallysomycin, pepleomycin and others which are further described herein.

Other DNA-modifying molecules may be used in targeted generecombination. For example, peptide nucleic acids may be used to inducemodifications to the genome of the target cell or cells (see, e.g.,Ecker, U.S. Pat. No. 5,986,053 herein incorporated by reference). Inbrief, synthetic nucleotides comprising, at least, a partial peptidebackbone are used to target a homologous genomic nucleotide sequence.Upon binding to the double-helical DNA, or through a mutagen ligated tothe peptide nucleic acid, modification of the target DNA sequence and/orrecombination is induced to take place. Targeting specificity isdetermined by the degree of sequence homology between the targetingsequence and the genomic sequence.

Furthermore, the present disclosure is not limited to the particularmethods which are used herein to execute modification of genomicsequences. Indeed, a number of methods are contemplated. For example,genes may be targeted using triple helix forming oligonucleotides (TFO).TFOs may be generated synthetically, for example, by PCR or by use of agene synthesizer apparatus. Additionally, TFOs may be isolated fromgenomic DNA if suitable natural sequences are found. TFOs may be used ina number of ways, including, for example, by tethering to a mutagen suchas, but not limited to, psoralen or chlorambucil (see, e.g., Havre etal., Proc Nat'l Acad Sci, U.S.A. 90:7879-7883, 1993; Havre et al., JVirol 67:7323-7331, 1993; Wang et al., Mol Cell Biol. 15:1759-1768,1995; Takasugi et al., Proc Nat'l Acad Sci, U.S.A. 88:5602-5606, 1991;Belousov et al., Nucleic Acids Res 25:3440-3444, 1997). Furthermore, forexample, TFOs may be tethered to donor duplex DNA (see, e.g., Chan etal., J Biol Chem 272:11541-11548, 1999). TFOs can also act by bindingwith sufficient affinity to provoke error-prone repair (Wang et al.,Science 271:802-805, 1996).

The methods disclosed herein are not necessarily limited to the natureor type of DNA-modifying reagent which is used. For example, suchDNA-modifying reagents release radicals which result in DNA strandbreakage. Alternatively, the reagents alkylate DNA to form adducts Whichwould block replication and transcription. In another alternative, thereagents generate crosslinks or molecules that inhibit cellular enzymesleading to strand breaks. Examples of DNA-modifying reagents which havebeen linked to oligonucleotides to form TFOs include, but are notlimited to, indolocarbazoles, napthalene diimide (NDI), transplatin,bleomycin, analogues of cyclopropapyrroloindole, andphenanthodihydrodioxins. In particular, indolocarbazoles aretopoisomerase I inhibitors. Inhibition of these enzymes results instrand breaks and DNA protein adduct formation (Arimondo el al.,Bioorganic and Medicinal Chem. 8, 777, 2000). NDI is a photooxidant thatcan oxidize guanines which could cause mutations at sites of guanineresidues (Nunez, et al., Biochemistry, 39, 6190, 2000). Transplatin hasbeen shown to react with DNA in a triplex target when the TFO is finkedto the reagent. This reaction causes the formation of DNA adducts whichwould be mutagenic (Columbier, et al., Nucleic Acids Research, 24: 4519,1996). Bleomycin is a DNA breaker, widely used as a radiation mimetic.It has been linked to oligonucleotides and shown to be active as abreaker in that format (Sergeyev, Nucleic Acids Research 23, 4400, 1995;Kane, et al., Biochemistry, 34, 16715, 1995). Analogues ofcyclopropapyrroloindole have been linked to TFOs and shown to alkylateDNA in a triplex target sequence. The alkylated DNA would then containchemical adducts which would be mutagenic (Lukhtanov, et al., NucleicAcids Research, 25, 5077, 1997). Phenanthodihydrodioxins are maskedquinones that release radical species upon photoactivation. They havebeen linked to TFOs and have been shown to introduce breaks into duplexDNA on photoactivation (Bendinskas et al., Bioconjugate Chem. 9, 555,1998).

Other methods of inducing modifications and/or recombination arecontemplated by the present disclosure. For example, another embodimentinvolves the induction of homologous recombination between an exogenousDNA fragment and the targeted gene (see e.g., Capecchi et al., Science244:1288-1292, 1989) or by using peptide nucleic acids (PNA) withaffinity for the targeted site. Still other methods include sequencespecific DNA recognition and targeting by polyamides (see e.g., Dervanet al., Curr (Ipin Chem Biol 3:688-693, 1999: Biochemistry 38:2143-2151,1999) and the use nucleases with site specific activity (e.2., zincfinger proteins, TALENs, Meganucleases and/or CRISPIRs).

The present disclosure is not limited to any particular frequency ofmodification and/or recombination. In some embodiments the methodsdisclosed herein result in a frequency of modification in the targetnucleotide sequence of from 0.2% to 3%. Nonetheless, any frequency(i.e., between 0% and 100%) of modification and/or recombination iscontemplated to he within the scope of the present disclosure. Thefrequency of modification and/or recombination is dependent on themethod used to induce the modification and/or recombination, the celltype used, the specific gene targeted and the DNA mutating reagent used,if any. Additionally, the method used to detect the modification and/orrecombination, due to limitations in the detection method, may notdetect all occurrences of modification and/or recombination.Furthermore, some modification and/or recombination events may besilent, giving no detectable indication that the modification and/orrecombination has taken place. The inability to detect silentmodification and/or recombination events gives an artificially lowestimate of modification and/or recombination. Because of these reasons,and others, the disclosure is not necessarily limited to any particularmodification and/or recombination frequency. In one embodiment, thefrequency of modification and/or recombination is between 0.01% and100%. In another embodiment, the frequency of modification and/orrecombination is between 0.01% and 50%. In yet another embodiment, thefrequency of modification and/or recombination is between 0.1% and 10%.in still yet another embodiment, the frequency of modification and/orrecombination is between 0.1% and 5%.

The term “frequency of mutation” as used herein in reference to apopulation of cells which are treated with a DNA-modifying molecule thatis capable of introducing a mutation into a target site in the cells'genome, refers to the number of cells in the treated population whichcontain the mutation at the target site as compared to the total numberof cells which are treated with the DNA-modifying molecule. For example,with respect to a population of cells which is treated with theDNA-modifying molecule TFO tethered to psoralen which is designed tointroduce a mutation at a target site in the cells' genome, a frequencyof mutation of 5% means that of a total of 100 cells which are treatedwith TFO-psoralen, 5 cells contain a mutation at the target site.

Although the present disclosure is not necessarily limited to any degreeof precision in the modification and/or recombination of DNA in thecell, it is contemplated that some embodiments of the present disclosurerequire higher degrees of precision, depending on the desired result.For example, the specific sequence changes required for gene repair(e.g., particular base changes) require a higher degree of precision ascompared to producing a gene knockout wherein only the disruption of thegene is necessary. With the methods of the present disclosure,achievement of higher levels of precision in modification and/orhomologous recombination techniques is greater than with prior artmethods.

Delivery of Gene Repair Oligonucleobases into Plant Cells

Any commonly known method used to transform a plant cell can be used fordelivering the gene repair oligonucleobases. Illustrative methods arelisted below. The methods and compositions herein may involve any ofmany methods to transfect the cells with the DNA-modifying reagent orreagents. Methods for the introduction of DNA modifying reagents into acell or cells are well known in the art and include, but are not limitedto, microinjection, electroporation, passive adsorption, calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, liposome fusion, lipofectin,nucleofection, protoplast fusion, retroviral infection, biolistics(i.e., particle bombardment) and the like.

The use of metallic microcarriers (microspheres) for introducing largefragments of DNA into plant cells having cellulose cell walls byprojectile penetration is well known to those skilled in the relevantart (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792and 5,204,253 describe general techniques for selecting microcarriersand devices for projecting them.

Specific conditions for using microcarriers in the methods disclosedherein may include the conditions desciibed international Publication WO99/07865. In an illustrative technique, ice cold microcarriers (60mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaCl₂ and 0.1 Mspermidine are added in that order; the mixture gently agitated, e.g.,by vortexing, for 10 minutes and then left at room temperature for 10minutes, whereupon the microcarriers are diluted in 5 volumes ofethanol, centrifuged and resuspended in 100% ethanol. Good results canbe obtained with a concentration in the adhering solution of 8-10microcarriers, 14-17 μg/mL mixed duplex oligonucleotide, 1.1-1.4 M CaCl₂and 18-22 mM spermidine. Optimal results were observed under theconditions of 8 μg/μL microcaniers, 16.5 μg/mL mixed duplexoligonucleotide, 1.3 M CaCl² and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into plant cellsusing microfibers to penetrate the cell wall and cell membrane. U.S.Pat. No. 5,302,523 to Coffee et al describes the use of silicon carbidefibers to facilitate transformation of suspension maize cultures ofBlack Mexican Sweet. Any mechanical technique that can be used tointroduce DNA for transformation of a plant cell using microfibers canbe used to deliver gene repair oligonucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repairoligonucleobase is as follows: Sterile microfibers (2 μg) are suspendedin 150 μL of plant culture medium containing about 10 μg of a mixedduplex oligonucleotide. A suspension culture is allowed to settle andequal volumes of packed cells and the sterile fiber/nucleotidesuspension are vortexed for 10 minutes and plated. Selective media areapplied immediately or with a delay of up to about 120 h as isappropriate for the particular trait.

In an alternative embodiment, the gene repair oligonucleobases can bedelivered to the plant cell by electroporation of a protoplast derivedfrom a plant part. The protoplasts are formed by enzymatic treatment ofa plant part, particularly a leaf, according to techniques well known tothose skilled in the art. See, e.g., Gallois et al, 1996, in Methods inMolecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al.,1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa,N.J. The protoplasts need not be cultured in growth media prior toelectroporation. Illustrative conditions for electroporation are 300,000protoplasts in a total volume of 0.3 mL with a concentration of generepair oligonucleobase of between 0.6-4 μg/mL.

In an alternative embodiment, nucleic acids are taken up by plantprotoplasts in the presence of the membrane-modifying agent polyethyleneglycol, according to techniques well known to those skilled in the art.In another alternative embodiment, the gene repair oligonucleobases canbe delivered by injecting it with a microcapillary into plant cells orinto protoplasts.

In an alternative embodiment, nucleic acids are embedded in microbeadscomposed of calcium alginate and taken up by plant protoplasts in thepresence of the membrane-modifying agent polyethylene glycol (see, e.g.,Sone et al., 2002, Liu et al., 2004).

In an alternative embodiment, nucleic acids frozen in water andintroduced into plant cells by bombardment in the form of microparticles(see, e.g., Gilmore, 1991, U.S. Pat. 5,219,746; Brinegar et al.).

In an alternative embodiment, nucleic acids attached to nanoparticlesare introduced into intact plant cells by incubation of the cells in asuspension containing the nanoparticie (see, e.g., Pasupathy et al.,2008) or by delivering them into intact cells through particlebombardment or into protoplasts by co-incubation (see, e.g., Torney etal., 2007).

In an alternative embodiment, nucleic acids complexed with penetratingpeptides and delivered into cells by co-incubation (see, e.g., Chugh etal., 2008, WO 2008148223 Al; Eudes and Chugh).

In an alternative embodiment, nucleic acids are introduced into intactcells through electroporation (see, e.g., He et al., 1998 US2003/0115641 A1, Dobres et al.).

In an alternative embodiment, nucleic acids are delivered into cells ofdry embryos by soaking them in a solution with nucleic acids (see, e.g.,Töpfer et al., 1989, Senaratna et al., 1991) or in other embodiments areintroduced by Cellsqueeze (SQZ Biotech).

Selection of Plants

In various embodiments, plants as disclosed herein can be of any speciesof dicotyledonous, monocotyledonous or gymnospermous plant, includingany woody plant species that grows as a tree or shrub, any herbaceousspecies, or any species that produces edible fruits, seeds orvegetables, or any species that produces colorful or aromatic flowers.For example, the plant maybe selected from a species of plant from thegroup consisting of canola, sunflower, corn, tobacco, sugar beet,cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum, tomato,mango, peach, apple, pear, strawberry, banana, melon, cassava, potato,carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea,field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin,cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus,triticale, alfalfa, rye, oats, turf and forage, grasses, flax, oilseedrape, mustard, cucumber, morning glory, balsam, pepper, eggplant,marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nutproducing plants insofar as they are not already specifically mentioned.

Plants and plant cells can be tested for resistance or tolerance to anherbicide using commonly known methods in the art, e.g., by growing theplant or plant cell in the presence of an herbicide and measuring therate of growth as compared to the growth rate in the absence of theherbicide.

As used herein, substantially normal growth of a plant, plant organ,plant tissue or plant cell is defined as a growth rate or rate of celldivision of the plant, plant organ, plant tissue, or plant cell that isat least 35%, at least 50%, at least 60%, or at least 75% of the growthrate or rate of cell division in a corresponding plant, plant organ,plant tissue or plant cell expressing the wild-type protein of interest.

As used herein, substantially normal development of a plant, plantorgan, plant tissue or plant cell is defined as the occurrence of one ormore development events in the plant, plant organ, plant tissue or plantcell that are substantially the same as those occurring in acorresponding plant, plant organ, plant tissue or plant cell expressingthe wild-type protein.

In certain embodiments plant organs provided herein include, but are notlimited to, leaves, stems, roots, vegetative buds, floral buds,meristems, embryos, cotyledons, endosperm, sepals, petals, pistils,carpels, stamens, anthers, macrospores, pollen, pollen tubes, ovules,ovaries and fruits, or sections, slices or discs taken therefrom Planttissues include, but are not limited to, callus tissues, ground tissues,vascular tissues, storage tissues, meristematic tissues, leaf tissues,shoot tissues, root tissues, gall tissues, plant tumor tissues, andreproductive tissues. Plant cells include, but are not limited to,isolated cells with cell walls, variously sized aggregates thereof, andprotoplasts.

Plants are substantially “tolerant” to a relevant herbicide when theyare subjected to it and provide a dose/response curve which is shiftedto the right when compared with that provided by similarly subjectednon-tolerant like plant. Such doseiresponse curves have “dose” plottedon the X-axis and “percentage kill”, “herbicidal effect”, etc., plottedon the y-axis. Tolerant plants will require more herbicide thannon-tolerant like plants in order to produce a given herbicidal effect.Plants that are substantially “resistant” to the herbicide exhibit few,if any, necrotic, lytic, chlorotic or other lesions, when subjected toherbicide at concentrations and rates which are typically employed bythe agrochemical community to kill weeds in the field. Plants which areresistant to an herbicide are also tolerant of the herbicide.

Generation of Plants

Tissue culture of various tissues of plant species and regeneration ofplants therefrom is known. For example, the propagation of a canolacultivar by tissue culture is described in any of the following but notlimited to any of the following: Chuong et al., “A Simple Culture Methodfor Brassica hypocotyls Protoplasts,” Plant Cell Reports 4:4-6, 1985;Barsby, T. L., et al., “A Rapid and Efficient Alternative Procedure forthe Regeneration of Plants from Hypocotyl Protoplasts of Brassicanapus,” Plant Cell Reports (Spring, 1996); Kartha, K., et al., “In vitroPlant Formation from Stem Explants of Rape,” Physiol. Plant, 31:217-220,1974; Narasimhulu, S., et al., “Species Specific Shoot RegenerationResponse of Cotyledonary Explants of Brassicas,” Plant Cell Reports(Spring 1988); Swanson, E., “Microspore Culture in Brassica,” Methods inMolecular Biology, Vol. 6, Chapter 17, p. 159, 1990.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybeans,” Crop Sci.31:333-337, 1991; Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. 82:633-635,1991; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113, 1992; Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports11:285-289, 1992; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5, 1992; and Shetty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251, 1992. Thedisclosures of U.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collinset al., and U.S. Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch etal., are hereby incorporated herein in their entirety by reference.

Exemplary Embodiments

In addition to the aspects and embodiments described and providedelsewhere in this disclosure, the following non-limiting list ofparticular embodiments are specifically contemplated.

-   1. A method of causing a genetic change in a cell, said method    comprising exposing said cell to a DNA cutter and a modified GRON.-   2. A cell comprising a DNA cutter and a GRON.-   3. The method or cell of any of the preceding embodiments, wherein    said cells is one or more species of cell selected from the group    consisting of plant, bacteria, yeast, fungi, algae, and mammalian.-   4. The method or cell of any of the preceding embodiments, wherein    said cells is one or more species of cell selected from the group    consisting of Escherichia coli, Mycobacterium smegmatis, Baccillus    subtilis, Chlorella, Bacillus thuringiensis, Saccharomyces    cerevisiae, Yarrowia lipolytica, Chlamydamonas rhienhardtii, Pichia    pastoris, Corynebacterium, Aspergillus niger, and Neurospora crassa.    Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza    sativa, Glycine max, Amaranthus tuberculatus, Linum usitatissimum,    and Zea mays-   5. The method or cell of any of the preceding embodiments, wherein    said cell is Yarrowia lipolytica.-   6. The method or cell of any of the preceding embodiments, wherein    said cell is a yeast cell that is not Saccharomyces cerevisiae.-   7. A method of causing a genetic change in a plant cell, said method    comprising exposing said cell to a DNA cutter and a modified GRON.-   8. A plant cell comprising a DNA cutter and a modified GRON.-   9. A method of causing a genetic change in a plant cell, said method    comprising exposing said cell to a DNA cutter and a GRON that    comprises DNA and RNA.-   10. A plant cell comprising a DNA cutter that comprises DNA and RNA.-   11. A method of causing a genetic change in a Acetyl-Coenzyme A    carboxylase (ACCase) gene in a cell, wherein said genetic change    causes a change in the Acetyl-Coenzyme A carboxylase (ACCase)    protein at one or more amino acid positions, said positions selected    from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and    2088 based on the numbering of the blackgrass reference sequence SEQ    ID NO:1 or at an analogous amino acid residue in an ACCase paralog    said method comprising exposing said cell to a modified GRON.-   12. A method of causing a genetic change in a Acetyl-Coenzyme A    carboxylase (ACCase) gene in a cell, wherein said genetic change    causes a change in the Acetyl-Coenzyme A carboxylase (ACCase)    protein at one or more amino acid positions, said positions selected    from the group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and    2088 based on the numbering of the blackgrass reference sequence SEQ    ID NO:1 or at an analogous amino acid residue in an ACCase paralog    said method comprising exposing said cell to a DNA cutter and a    modified GRON.-   13. A method for producing a plant or plant cell, comprising    introducing into a plant cell a gene repair oligonucleobase (GRON)    with a targeted mutation in an Acetyl-Coenzyme A carboxylase    (ACCase) gene to produce a plant cell with an ACCase gene that    expresses an ACCase protein comprising a mutation at one or more    amino acid positions corresponding to a position selected from the    group consisting of 1781, 1783, 1786, 2078, 2079, 2080 and 2088    based on the numbering of the blackgrass reference sequence SEQ ID    NO:1 or at an analogous amino acid residue in an ACCase paralog.-   14. A method for producing a plant or plant cell, comprising    introducing into a plant cell a DNA cutter and a gene repair    oligonucleobase (GRON) with a targeted mutation in an    Acetyl-Coenzyme A carboxylase (ACCase) gene to produce a plant cell    with an ACCase gene that expresses an ACCase protein comprising a    mutation at one or more amino acid positions corresponding to a    position selected from the group consisting of 1781, 1783, 1786,    2078, 2079, 2080 and 2088 based on the numbering of the blackgrass    reference sequence SEQ ID NO:1 or at an analogous amino acid residue    in an ACCase paralog.-   15. A fertile plant comprising an Acetyl-Coenzyme A carboxylase    (ACCase) gene that encodes a protein comprising a mutation at    position 2078 based on the numbering of the blackgrass reference    sequence SEQ ID NO:1 or at an analogous amino acid residue in an    ACCase paralog.-   16. A fertile rice plant comprising an Acetyl-Coenzyme A carboxylase    (ACCase) gene that encodes a protein comprising a mutation at    position 2078 based on the numbering of the blackgrass reference    sequence SEQ ID NO:1 or at an analogous amino acid residue in an    ACCase paralog.-   17. A plant cell comprising an Acetyl-Coenzyme A carboxylase    (ACCase) gene that encodes a protein comprising a mutation at    position 2078 based on the numbering of the blackgrass reference    sequence SEQ ID NO:1 or at an analogous amino acid residue in an    ACCase paralog and that further comprises an Acetyl-Coenzyme A    carboxylase (ACCase) gene that encodes a protein comprising a    mutation at one or more amino acid positions, said positions    selected from the group consisting of 1781, 1783, 1786, 2079, 2080    and 2088 based on the numbering of the blackgrass reference sequence    SEQ ID NO:1 or at an analogous amino acid residue in an ACCase    paralog.-   18. A fertile plant comprising an Acetyl-Coenzyme A carboxylase    (ACCase) gene that encodes a protein comprising a mutation at    position 2078 based on the numbering of the blackgrass reference    sequence SEQ ID NO:1 or at an analogous amino acid residue in an    ACCase paralog and that further comprises an Acetyl-Coenzyme A    carboxylase (ACCase) gene that encodes a protein comprising a    mutation at one or more amino acid positions, said positions    selected from the group consisting of 1781, 1783, 1786, 2079, 2080    and 2088 based on the numbering of the blackgrass reference sequence    SEQ ID NO:1 or at an analogous amino acid residue in an ACCase    paralog.-   19. A method of causing a genetic change in a Acetyl-Coenzyme A    carboxylase (ACCase) gene in a cell, wherein said genetic change    causes a change in the Acetyl-Coenzyme A carboxylase (ACCase)    protein at position 2078 based on the numbering of the blackgrass    reference sequence SEQ ID NO:1 or at an analogous amino acid residue    in an ACCase paralog said method comprising exposing said cell to a    modified GRON.-   20. A method of causing a genetic change in a Acetyl-Coenzyme A    carboxylase (ACCase) gene in a cell, wherein said genetic change    causes a change in the Acetyl-Coenzyme A carboxylase (ACCase)    protein at position 2078 based on the numbering of the blackgrass    reference sequence SEQ ID NO:1 or at an analogous amino acid residue    in an ACCase paralog said method comprising exposing said cell to a    DNA cutter and a modified GRON.-   21. The method, plant or cell of any of the preceding embodiments,    wherein said mutation or change in an Acetyl-Coenzyme A carboxylase    (ACCase) gene, if present results in an Acetyl-Coenzyme A    carboxylase (ACCase) protein comprising one or more selected from    the group consisting of an isoleucine to alanine at a position    corresponding to position 1781 of SEQ ID NO:1; an isoleucine to    leucine at a position corresponding to position 1781 of SEQ ID NO:1;    an isoleucine to methionine at a position corresponding to position    1781 of SEQ ID NO:1; an isoleucine to asparagine at a position    corresponding to position 1781 of SEQ ID NO:1; an isoleucine to    serine at a position corresponding to position 1781 of SEQ ID NO:1;    an isoleucine to threonine at a position corresponding to position    1781 of SEQ ID NO:1; an isoleucine to valine at a position    corresponding to position 1781 of SEQ ID NO:1; a glycine to cysteine    at a position corresponding to position 1783 of SEQ ID NO:1; an    alanine to proline at a position corresponding to position 1786 of    SEQ ID NO:1; an aspartate to glycine at a position corresponding to    position 2078 of SEQ ID NO:1; an aspartate to lysine at a position    corresponding to position 2078 of SEQ ID NO:1; an aspartate to    threonine at a position corresponding to position 2078 of SEQ ID    NO:1; a serine to phenylalanine at a position corresponding to    position 2079 of SEQ ID NO:1; a lysine to glutamate at a position    corresponding to position 2080 of SEQ ID NO:1; a cysteine to    phenylalanine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to glycine at a position corresponding to position    2088 of SEQ ID NO:1; a cysteine to histidine at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to lysine    at a position corresponding to position 2088 of SEQ ID NO:1; a    cysteine to leucine at a position corresponding to position 2088 of    SEQ ID NO:1; a cysteine to asparagine at a position corresponding to    position 2088 of SEQ ID NO:1; a cysteine to proline at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to    glutamine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to arginine at a position corresponding to position    2088 of SEQ ID NO:1; a cysteine to serine at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to    threonine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to valine at a position corresponding to position    2088 of SEQ ID NO:1; and a cysteine to a tryptophan at a position    corresponding to position 2088 of SEQ ID NO:1.-   22. The plant or cell of any of the preceding embodiments, or a    plant or plant cell made by any of the methods of the preceding    embodiments, wherein said plant or cell comprises an Acetyl-Coenzyme    A carboxylase (ACCase) protein comprising one or more selected from    the group consisting of an isoleucine to alanine at a position    corresponding to position 1781 of SEQ ID NO:1; an isoleucine to    leucine at a position corresponding to position 1781 of SEQ ID NO:1;    an isoleucine to methionine at a position corresponding to position    1781 of SEQ ID NO:1; an isoleucine to asparagine at a position    corresponding to position 1781 of SEQ ID NO:1; an isoleucine to    serine at a position corresponding to position 1781 of SEQ ID NO:1;    an isoleucine to threonine at a position corresponding to position    1781 of SEQ ID NO:1; an isoleucine to valine at a position    corresponding to position 1781 of SEQ ID NO:1; a glycine to cysteine    at a position corresponding to position 1783 of SEQ ID NO:1; an    alanine to proline at a position corresponding to position 1786 of    SEQ ID NO:1; an aspartate to glycine at a position corresponding to    position 2078 of SEQ ID NO:1; an aspartate to lysine at a position    corresponding to position 2078 of SEQ ID NO:1; an aspartate to    threonine at a position corresponding to position 2078 of SEQ ID    NO:1; a serine to phenylalanine at a position corresponding to    position 2079 of SEQ ID NO:1; a lysine to glutamate at a position    corresponding to position 2080 of SEQ ID NO:1; a cysteine to    phenylalanine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to glycine at a position corresponding to position    2088 of SEQ ID NO:1; a cysteine to histidine at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to lysine    at a position corresponding to position 2088 of SEQ ID NO:1; a    cysteine to leucine at a position corresponding to position 2088 of    SEQ ID NO:1; a cysteine to asparagine at a position corresponding to    position 2088 of SEQ ID NO:1; a cysteine to proline at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to    glutamine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to arginine at a position corresponding to position    2088 of SEQ ID NO:1; a cysteine to serine at a position    corresponding to position 2088 of SEQ ID NO:1; a cysteine to    threonine at a position corresponding to position 2088 of SEQ ID    NO:1; a cysteine to valine at a position corresponding to position    2088 of SEQ ID NO:1; and a cysteine to a tryptophan at a position    corresponding to position 2088 of SEQ ID NO:1.-   23. The plant or cell of any of the preceding embodiments, or a    plant or cell made by any of the methods of the preceding    embodiments, wherein said plant or plant cell comprises an    Acetyl-Coenzyme A carboxylase (ACCase) gene that encodes a protein    comprising a mutation at one or more amino acid positions, said    positions selected from the group consisting of 1781, 1783, 1786,    2078, 2079, 2080 and 2088 based on the numbering of the blackgrass    reference sequence SEQ ID NO:1 or at an analogous amino acid residue    in an ACCase paralog.-   24. The plant or cell of any of the preceding embodiments, or a    plant or cell made by any of the methods of the preceding    embodiments, wherein said plant or cell comprises an Acetyl-Coenzyme    A carboxylase (ACCase) gene that encodes a protein comprising a    mutation at position 2078 based on the numbering of the blackgrass    reference sequence SEQ ID NO:1 or at an analogous amino acid residue    in an ACCase paralog and that further comprises an Acetyl-Coenzyme A    carboxylase (ACCase) gene that encodes a protein comprising a    mutation at one or more amino acid positions, said positions    selected from the group consisting of 1781, 1783, 1786, 2079, 2080    and 2088 based on the numbering of the blackgrass reference sequence    SEQ ID NO:1 or at an analogous amino acid residue in an ACCase    paralog.

In each of the foregoing ACCase embodiments 11-24, whether methods,plants, cells, or otherwise, the following are suitable mutations foruse therein:

Amino Amino Acid Acid Change Codon Change Change Codon Change I1781AATA > GCT C2088F TGC > TTT ATA > GCC TGC > TTC ATA > GCA ATA > GCGC2088G TGC > GGT TGC > GGC I1781L ATA > CTT TGC > GGA ATA > CTC TGC >GGG ATA > CTA ATA > CTG C2088H TGC > CAT ATA > TTA TGC > CAC ATA > TTGC2088K TGC > AAA I1781M ATA > ATG TGC > AAG I1781N ATA > AAT C2088LTGC > CTT ATA > AAC TGC > CTC TGC > CTA I1781S ATA > TCT TGC > CTG ATA >TCC TGC > TTA ATA > TCA TGC > TTG ATA > TCG C2088N TGC > AAT I1781TATA > ACT TGC > AAC ATA > ACC ATA > ACA C2088P TGC > CCT ATA > ACG TGC >CCC TGC > CCA I1781V ATA > GTT TGC > CCG ATA > GTC ATA > GTA C2088QTGC > CAA ATA > GTG TGC > CAG G1783C GGA > TGT C2088R TGC > CGT GGA >TGC TGC > CGC TGC > CGA A1786P GCT > CCT TGC > CGG GCT > CCC TGC > AGAGCT > CCA TGC > AGG GCT > CCG C2088S TGC > TCT D2078G GAT > GGT TGC >TCC GAT > GGC TGC > TCA GAT > GGA TGC > TCG GAT > GGG C2088T TGC > ACTD2078K GAT > AAA TGC > ACC GAT > AAG TGC > ACA TGC > ACG D2078T GAT >ACT GAT > ACC C2088V TGC > GTT GAT > ACA TGC > GTC GAT > ACG TGC > GTATGC > GTG S2079F AGC > TTT AGC > TTC C2088W TGC > TGG K2080E AAG > GAAAAG > GAG

Alternative mutations include, but are not limited to, the following:

S2079A AGC > GCT AGC > GCC AGC > GCA AGC > GCG G1783A GGA > GCT GGA >GCC GGA > GCA GGA > GCG A1786G GCT > GGT GCT > GGC GCT > GGA GCT > GGG

With regard to embodiments 11-24, corresponding positions to 1781m 1783,1786, 2078, 2079, and 2080 based on the numbering of the blackgrassreference sequence are well known in the art and readily obtainable fromappropriate sequence databases. By way of example, the following tableshows the corresponding positions in the rice ACCase sequence:

Am OsI OsJ I1781 I1792 I1779 G1783 G1794 G1781 A1786 A1797 A1784 D2078D2089 D2076 S2079 S2090 S2077 K2080 K2091 K2078 C2088 C2099 C2086 Am:Alopecurus myosuroide; OsI: Oryza sativa indica variety; OsJ: Oryzasativa japonica variety

-   25. A method for producing a plant or plant cell with a mutated    EPSPS gene, comprising introducing into a plant cell a gene repair    oligonucleobase (GRON) with a targeted mutation in an 5-enol    pyruvylshikimate-3-phosphate synthase (EPSPS) gene to produce a    plant cell with an EPSPS gene that expresses an EPSPS protein    comprising a mutation at one or more amino acid positions    corresponding to a position selected from the group consisting of    96, 97 and 101 based on the numbering of the amino acid sequence for    the Escherichia coli reference sequence SEQ ID NO:2 or at an    analogous amino acid residue in an EPSPS paralog.-   26. A method for producing a plant or plant cell with a mutated    EPSPS gene, comprising introducing into a plant cell a DNA cutter    and a gene repair oligonucleobase (GRON) with a targeted mutation in    an 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) gene to    produce a plant cell with an EPSPS gene that expresses an EPSPS    protein comprising a mutation at one or more amino acid positions    corresponding to a position selected from the group consisting of    96, 97 and 101 based on the numbering of the amino acid sequence for    the Escherichia coli reference sequence SEQ ID NO:2 or at an    analogous amino acid residue in an EPSPS paralog.-   27. A plant or cell with a mutated EPSPS gene, wherein said plant or    cell is made by a method introducing into a plant cell a DNA cutter    and a gene repair oligonucleobase (GRON) with a targeted mutation in    an 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) gene to    produce a plant cell with an EPSPS gene that expresses an EPSPS    protein comprising a mutation at one or more amino acid positions    corresponding to a position selected from the group consisting of    96, 97 and 101 based on the numbering of the amino acid sequence for    the Escherichia coli reference sequence SEQ ID NO:2 or at an    analogous amino acid residue in an EPSPS paralog.-   28. The plant or cell of any of the preceding embodiments, or a    plant or cell made by any of the methods of the preceding    embodiments, wherein the plant or plant cell expresses an EPSPS    protein comprising a mutation at one or more amino acid positions    are selected from the group consisting of a glycine to alanine at a    position corresponding to position 96 of SEQ ID NO:2; a threonine to    isoleucine at a position corresponding to position 97 of SEQ ID    NO:2; a proline to alanine at a position corresponding to position    101 of SEQ ID NO:2; a proline to serine at a position corresponding    to position 101 of SEQ ID NO:2; and a proline to threonine at a    position corresponding to position 101 of SEQ ID NO:2.-   29. The plant or cell of any of the preceding embodiments, or a    plant or cell made by any of the methods of the preceding    embodiments, wherein the plant or plant cell expresses an EPSPS    protein comprising mutation combinations selected from the group    consisting of a threonine to isoleucine at a position corresponding    to position 97 of SEQ ID NO:2 and a proline to alanine at a position    corresponding to position 101 of SEQ ID NO:2; a threonine to    isoleucine at a position corresponding to position 97 of SEQ ID    NO:2and a proline to alanine at a position corresponding to position    101 of SEQ ID NO:2; a threonine to isoleucine at a position    corresponding to position 97 of SEQ ID NO:2 and a proline to serine    at a position corresponding to position 101 of SEQ ID NO:2; and a    threonine to isoleucine at a position corresponding to position 97    of SEQ ID NO:2 and a proline to threonine at a position    corresponding to position 101 of SEQ ID NO:2.

With regard to embodiments 25-30, corresponding positions to 96, 97 and101 based on the numbering of the Escherichia coli reference sequenceSEQ ID NO:2 are well known in the art and readily obtainable fromappropriate sequence databases. See e.g., U.S. Pat. No. 8,268,622. Byway of example, the following table shows the corresponding positions inthe flax EPSPS sequence:

Flax EPSPS E. coli EPSPS Gene1 Gene2 G96 G176 G177 T97 T177 T178 P101P181 P182

E. coli EPSPS sequence is AroA having the sequence

(SEQ ID NO: 10) MESLTLQPIARVDGTINLPGSKTVSNRALLLAALAHGKTVLTNLLDSDDVRHMLNALTALGVSYTLSADRTRCEIIGNGGPLHAEGALELFLGNAGTAMRPLAAALCLGSNDIVLTGEPRMKERPIGHLVDALRLGGAKITYLEQENYPPLRLQGGFTGGNVDVDGSVSSQFLTALLMTAPLAPEDTVIRIKGDLVSKPYIDITLNLMKTFGVEIENQHYQQFVVKGGQSYQSPGTYLVEGDASSASYFLAAAAIKGGTVKVTGIGRNSMQGDIRFADVLEKMGATICWGDDYISCTRGELNAIDMDMNHIPDAAMTIATAALFAKGTTRLRNIYNWRVKETDRLFAMATELRKVGAEVEEGHDYIRITPPEKLNFAEIATYNDHRMAMCFSLVALSDTPVTILDPKCTAKTFPDYFEQLARISQAA

Flax gene 1 sequence is lcl-g41452_1333 having the sequence

(SEQ ID NO: 11) MALVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRRSGNVAAAAAAPLRVSASLTTAAEKASTVPEEVVLQPIKDISGIVTLPGSKSLSNRILLLAALSEGTTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGCGGVFPVGKLAKNDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQLGADVTCSSTSCPPVHVNGQGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEIEIVDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEVLEKMGAKVIWTENSVTVTGPPRDASGRKHLRAVDVNMNKMPDVAMTLAVVALYADGPTAIRDVASWRVKETERMIAICTELRKLGATVEEGPDYCIITPPEKLNIAEIDTYDDHRMAMAFSLAACADVPVTIRDPGC TKKTFPDYFEVLERYTKH

Flax gene 2 sequence is lcl-g40547_1271 having the sequence

(SEQ ID NO: 12) MAQVTKICGGANAVALPATFGTRRTKSISSSVSFRSSTSPPSLKQRRLLGNVAAAAAAAPLRISASLATAAEKASTVPEEIVLQPIKDISGIVTLPGSKSLSNRILLLAALSEGKTVVDNLLNSDDVHYMLGALKTLGLNVEHSSEQKRAIVEGRGGVFPVGKLGKNDIELFLGNAGTAMRPLTAAVTAAGGNSSYILDGVPRMRERPIGDLVVGLKQLGADVSCSSTSCPPVHVNAKGGLPGGKVKLSGSISSQYLTALLMAAPLALGDVEIEIVDKLISVPYVDMTLKLMERFGVAVEHSGSWDRFFVKGGQKYKSPGNAYVEGDASSASYFLAGAAITGGTITVEGCGTSSLQGDVKFAEVLEKMGAKVTWTETSVTVTGPPRDASGKKHLRAVDVNMNKMPDVAMTLAVVALYADGPTAIRDVASWRVKETERMIAVCTELRKLGATVEEGPDYCIITPPEKLSIAEIDTYDDHRMAMAFSLAACADVPVTIRDPG CTKKTFPDYFEVLERYTKH

-   30. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is one or more selected from a CRISPR, a TALEN, a    zinc finger, meganuclease, and a DNA-cutting antibiotic.-   31. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is a CRISPR or a TALEN.-   32. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is a CRISPR.-   33. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is a TALEN.-   34. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is one or more DNA-cutting antibiotics selected from    the group consisting of bleomycin, zeocin, phleomycin, tallysomycin    and pepleomycin.-   35. The method or cell of any of the preceding embodiments, wherein    said DNA cutter is zeocin.-   36. The method or cell of any of the preceding embodiments, wherein    said GRON is single stranded.-   37. The method or cell of any of the preceding embodiments, wherein    the GRON is a chemically protected oligonucleotide.-   38. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a chemically protected oligonucleotide protected    at the 5′ end.-   39. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a chemically protected oligonucleotide protected    at the 3′ end.-   40. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a chemically protected oligonucleotide protected    at the 5′ and 3′ ends.-   41. The method or cell of any of the preceding embodiments, wherein    the GRON comprises one or more selected from a Cy3 group, a 3PS    group, and a 2′-O-methyl group.-   42. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a Cy3 group.-   43. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a Cy3 group at the first base on the 5′ end.-   44. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a Cy3 group at the first base on the 3′ end.-   45. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 3PS group.-   46. The method or cell of any of the preceding embodiments, wherein    the GRON comprises two or more 3PS groups.-   47. The method or cell of any of the preceding embodiments, wherein    the GRON comprises three or more 3PS groups.-   48. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 3PS group at the first base on the 5′ end.-   49. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 3PS group at the first base on the 3′ end.-   50. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group.-   51. The method or cell of any of the preceding embodiments, wherein    the GRON comprises two or more 2′-O-methyl groups.-   52. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group at the first base on the 5′    end.-   53. The method or cell of any of the preceding embodiments, wherein    the GRON has a 2′-O-methyl group at the first base on the 5′ end and    does not have any other 2′-O-methyl groups.-   54. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first two or    more bases at the 5′ end.-   55. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first three or    more bases at the 5′ end.-   56. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first four or    more bases at the 5′ end.-   57. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first five or    more bases at the 5′ end.-   58. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first six or    more bases at the 5′ end.-   59. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first seven or    more bases at the 5′ end.-   60. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the eight four or    more bases at the 5′ end.-   61. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first nine or    more bases at the 5′ end.-   62. The method or cell of any of the preceding embodiments, wherein    the GRON comprises a 2′-O-methyl group on each of the first ten or    more bases at the 5′ end.-   63. The method or cell of any of the preceding embodiments, wherein    the GRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA base at the    5′ end.-   64. The method or cell of any of the preceding embodiments, wherein    said GRON has a wobble base pair relative to the target sequence for    the genetic change.-   65. The method or cell of any of the preceding embodiments, wherein    said GRON is between 15 and 60 nucleotides in length.-   66. The method or cell of any of the preceding embodiments, wherein    said GRON is 41 nucleotides in length.-   67. The method or cell of any of the preceding embodiments, wherein    said GRON is between 50 and 110 nucleotides in length.-   68. The method or cell of any of the preceding embodiments, wherein    said GRON is 101 nucleotides in length.-   69. The method or cell of any of the preceding embodiments, wherein    said GRON is between 150 and 210 nucleotides in length.-   70. The method or cell of any of the preceding embodiments, wherein    said GRON is 201 nucleotides in length.-   71. The method or cell of any of the preceding embodiments, wherein    said GRON is between 70 and 210 nucleotides in length.-   72. The method or cell of any of the preceding emodiments, wherein    said GRON is longer than 70 nucleotides in length.-   73. The method or cell of any of the preceding emodiments, wherein    said GRON is longer than 100 nucleotides in length.-   74. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 165 nucleotides in length.-   75. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 175 nucleotides in length.-   76. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 185 nucleotides in length.-   77. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 195 nucleotides in length.-   78. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 200 nucleotides in length.-   79. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 210 nucleotides in length.-   80. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 220 nucleotides in length.-   81. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 230 nucleotides in length.-   82. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 240 nucleotides in length.-   83. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 250 nucleotides in length.-   84. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 260 nucleotides in length.-   85. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 270 nucleotides in length.-   86. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 280 nucleotides in length.-   87. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 290 nucleotides in length.-   88. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 300 nucleotides in length.-   89. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 400 nucleotides in length.-   90. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 500 nucleotides in length.-   91. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 600 nucleotides in length.-   92. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 700 nucleotides in length.-   93. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 800 nucleotides in length.-   94. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 900 nucleotides in length.-   95. The method or cell of any of the preceding embodiments, wherein    said GRON is longer than 1000 nucleotides in length.-   96. The method or cell of any of the preceding embodiments wherein    said plant is selected from the group consisting of canola,    sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley,    rice, alfalfa, barley, sorghum, tomato, mango, peach, apple, pear,    strawberry, banana, melon, cassava, potato, carrot, lettuce, onion,    soy bean, soya spp, sugar cane, pea, chickpea, field pea, fava bean,    lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower,    kale, field beans, poplar, pine, eucalyptus, grape, citrus,    triticale, alfalfa, rye, oats, turf and forage grasses, flax,    oilseed rape, mustard, cucumber, morning glory, balsam, pepper,    eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris,    and lily.-   97. The method or cell of any of the preceding embodiments wherein    said plant is canola.-   98. The method or cell of any of the preceding embodiments wherein    said plant is corn-   99. The method or cell of any of the preceding embodiments wherein    said plant is maize.-   100. The method or cell of any of the preceding embodiments wherein    said plant is rice.-   101. The method or cell of any of the preceding embodiments wherein    said plant is sorghum.-   102. The method or cell of any of the preceding embodiments wherein    said plant is potato.-   103. The method or cell of any of the preceding embodiments wherein    said plant is soy bean.-   104. The method or cell of any of the preceding embodiments wherein    said plant is flax.-   105. The method or cell of any of the preceding embodiments wherein    said plant is oilseed rape.-   106. The method or cell of any of the preceding embodiments wherein    said plant is cassava.-   107. The method or cell of any of the preceding embodiments wherein    said plant is sunflower.-   108. A method of causing a genetic change in a plant cell, said    method comprising exposing said cell to a CRISPR and a modified    GRON.-   109. The method or cell of any of the preceding embodiments wherein    multiple genetic changes are made.-   110. The method or cell of any of the preceding embodiments wherein    two or more guide RNAs are used.-   111. The method or cell of any of the preceding embodiments wherein    each of the more than one guide RNAs is complimentary to a different    target for genetic change.-   112. The method or cell of any of the preceding embodiments wherein    the CRISPR includes a nickase.-   113. The method or cell of any of the preceding embodiments wherein    the DNA cutter includes two or more nickases.-   114. The method or cell of any of the preceding embodiments wherein    two or more nickases cuts on opposite strands of the target nucleic    acid sequence.-   115. The method or cell of any of the preceding embodiments wherein    two or more nickases cuts on the same strand of the target nucleic    acid sequence.-   116. A non-transgenic herbicide resistant or tolerant plant made by    the method or from the cell of one any of the preceding embodiments.-   117. The method or cell of any of the preceding embodiments, wherein    said plant cell has a genetic change or mutation in Acetyl-Coenzyme    A carboxylase (ACCase) and is selected from the group consisting of    barley, maize, millet, oats, rye, rice, sorghum, sugarcane, turf    grasses, and wheat.-   118. The method or cell of any of the preceding embodiments, wherein    said plant cell has a genetic change or mutation in Acetyl-Coenzyme    A carboxylase (ACCase) and is resistant or tolerant to one or more    herbicides.-   119. The method or cell of any of the preceding embodiments, wherein    said plant cell has a genetic change or mutation in Acetyl-Coenzyme    A carboxylase (ACCase), is resistant to one or more    ACCase-inhibiting herbicides.-   120. The method or cell of any of the preceding embodiments, wherein    said plant cell has a genetic change or mutation in Acetyl-Coenzyme    A carboxylase (ACCase), is resistant to one or more herbicides    selected from the group consisting of alloxydim, butroxydim,    clethodim, cloproxydim, cycloxydim, sethoxydim, tepraloxydim,    tralkoxydim, chlorazifop, clodinafop, clofop, diclofop, fenoxaprop,    fenoxaprop-P, fenthiaprop, fluazifop, fluazifop-P, haloxyfop,    haloxyfop-P, isoxapyrifop, propaquizafop, quizalofop, quizalofop-P,    trifop, pinoxaden, agronomically acceptable salts and esters of any    of these herbicides, and combinations thereof.-   121. The method or cell of any of the preceding embodiments, wherein    said plant cell has a genetic change or mutation in    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein    said plant cell is selected from the group consisting of corn,    wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton,    sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato,    tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce,    peas, lentils, grape and turf grasses.-   122. The method or cell of any of the preceding embodiments, wherein    said plant or plant cell has a genetic change or mutation in    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein    plant or plant cell is resistant to at least one herbicide.-   123. The method or cell of any of the preceding embodiments, wherein    said plant or plant cell has a genetic change or mutation in    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein    plant or plant cell is resistant to a herbicide of the    phosphonomethylglycine family.-   124. The method or cell of any of the preceding embodiments, wherein    said plant or plant cell has a genetic change or mutation in    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein    plant or plant cell is resistant to glyphosate.-   125. The method or cell of any of the preceding embodiments, wherein    said plant or plant cell has a genetic change or mutation in    5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein    plant or plant cell is selected from the group consisting of corn,    wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton,    sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato,    tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce,    peas, lentils, grape and turf grasses.-   126. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at one allele of    the gene.-   127. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at two alleles of    the gene.-   128. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at three alleles    of the gene.-   129. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at four alleles of    the gene.-   130. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at one, two,    three, four, five, six, seven, eight, nine, ten, eleven, or twelve    alleles of the gene.-   131. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell comprises a deletion or    insertion resulting in a knockout of one allele of the gene.-   132. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell comprises a deletion or    insertion resulting in a knockout of two alleles of the gene.-   133. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell comprises a deletion or    insertion resulting in a knockout of three alleles of the gene.-   134. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell comprises a deletion or    insertion resulting in a knockout of four alleles of the gene.-   135. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell comprises a deletion or    insertion resulting in a knockout of one, two, three, four, five,    six, seven, eight, nine, ten, eleven, or twelve alleles of the gene.-   136. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at one allele of    the gene and a second allele of the gene comprises a deletion or    insertion resulting in a knockout of said second allele.-   137. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at one allele of    the gene and a second allele and third allele of the gene comprises    a deletion or insertion resulting in a knockout of said second    allele and said third allele.-   138. The method or cell of any of the preceding embodiments, wherein    the genetic change or mutation in the cell occurs at one allele of    the gene and a second allele, third allele, and fourth allele of the    gene comprises a deletion or insertion resulting in a knockout of    said second allele, said third allele and said fourth allele.-   139. The method or cell of any of the preceding embodiments, wherein    the genetic change in the cell comprises at least one mutation at    one allele and at least one knockout in another allele.-   140. The method or cell of any of the preceding embodiments, wherein    the genetic change in the cell comprises at least one mutation at    one allele and at least one knockout in at least one other allele.-   141. The method or cell of any of the preceding embodiments, wherein    the genetic change in the cell comprises at least one mutation at    one allele and at least one knockout in at least two other alleles.-   142. The method or cell of any of the preceding embodiments, wherein    the genetic change in the cell comprises at least one mutation at    one allele and at least one knockout in at least three other    alleles.-   143. The method or cell of any of the preceding embodiments, wherein    the genetic change in the cell comprises at least one mutation at    one allele and a knockout in all other alleles.

EXAMPLES

The following are examples, which illustrate procedures for practicingthe methods and compositions described herein. These examples should notbe construed as limiting.

Example 1: GRON Length

Sommer et al., (Mol Biotechnol. 33:115-22, 2006) describes a reportersystem for the detection of in vivo gene conversion which relies upon asingle nucleotide change to convert between blue and green fluorescencein green fluorescent protein (GFP) variants. This reporter system wasadapted for use in the following experiments using Arabidopsis thalianaas a model species in order to assess efficiency of GRON conversionfollowing modification of the GRON length.

In short, for this and the subsequent examples an Arabidopsis thalianaline with multiple copies of a blue fluorescent protein gene was createdby methods known to those skilled in the art (see, e.g., Clough andBrent, 1998). Root-derived meristematic tissue cultures were establishedwith this line, which was used for protoplast isolation and culture(see, e.g., Mathur et al., 1995). GRON delivery into protoplasts wasachieved through polyethylene glycol (PEG) mediated GRON uptake intoprotoplasts. A method using a 96-well format, similar to that describedby similar to that described by Fujiwara and Kato (2007) was used. Inthe following the protocol is briefly described. The volumes given arethose applied to individual wells of a 96-well dish.

-   -   1. Mix 6.25 μl of GRON (80 μM) with 25 μl of Arabidopsis        thaliana BFP transgenic root meristematic tissue-derived        protoplasts at 5×10⁶ cells/ml in each well of a 96 well plate.    -   2. 31.25 μl of a 40% PEG solution was added and the protoplasts        were mixed.    -   3. Treated cells were incubated on ice for 30 min.    -   4. To each well 200 μl of W5 solution was added and the cells        mixed.    -   5. The plates were allowed to incubate on ice for 30 min        allowing the protoplasts to settle to the bottom of each well.    -   6. 200 μl of the medium above the settled protoplasts was        removed.    -   7. 85 μl of culture medium (MSAP, see Mathur et al., 1995) was        added.    -   8. The plates were incubated at room temperate in the dark for        48 hours. The final concentration of GRON after adding culture        medium is 8 μM.

Forty eight hours after GRON delivery samples were analyzed by flowcytometry in order to detect protoplasts whose green and yellowfluorescence is different from that of control protoplasts (BFPOindicates non-targeting GRONs with no change compared to the BFP target;C is the coding strand design and NC is the non-coding strand design). Asingle C to T nucleotide difference (coding strand) or G to A nucleotidetargeted mutation (non-coding strand) in the center of the BFP4molecules. The green fluorescence is caused by the introduction of atargeted mutation in the BFP gene, resulting in the synthesis of GFP.The results are shown in FIG. 1.

Table 2 shows the sequences of exemplary 101-mer and 201-mer BFP4/NC5′-3PS/3′-3PS GRONs designed for the conversion of a blue fluorescentprotein (BFP) gene to green fluorescence. “3PS” denotes 3 phosphothioatelinkages at each of the 5′ and 3′ oligo ends.

TABLE 2 Exemplary GRON Nucleotide Sequences for BFP to GFP conversionGKON GKON Nucleotide Name Sequence BFP4/NC G* T*C*G TGC TGC TTC ATG101-mer TGG TCG GGG TAG CGG CTG AAG CAC TGC ACG CCG TAGGTG AAG GTG GTC ACG AGG GTG GGC CAG GGC ACG GGC AGC TTG CCG G*T*G* G(SEQ ID NO: 13) BFP0/NC G* T*C*G TGC TGC TTC 101-mer ATG TGG TCG GGG TAGCGG CTG AAG CAC TGC ACG CCG TGG GTG AAG GTG GTC ACG AGG GTGGGC CAG GGC ACG GGC AGC TTG CCG G*T*G *G (SEQ ID NO: 14) BFP4/CC *C*A*C CGG CAA GCT 101-mer GCC CGT GCC CTG GCC CAC CCT CGT GAC CACCTT CAC CTA CGG CGT GCA GTG CTT CAG CCG CTA CCC CGA CCA CATGAA GCA GCA C*G*A *C (SEQ ID NO: 15) BFP0/C C*C*A*CCGGCAAGCTGCCCG101-mer TGCCCTGGCCCACCCTCGTGA CCACCTTCACCCACGGCGTGCAGTGCTTCAGCCGCTACCCCG ACCACATGAAGCAGCAC*G*A* C (SEQ ID NO: 16) BFP4/NCA*A*G*ATGGTGCGCTCCTGGA 201-mer CGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTC ATGTGGTCTGGGTAGCGGCTGA AGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAG GGCACGGGCAGCTTGCCGGTGG TGCAGATGAACTTCAGGGTCAGCTTGCCG TAGGTGGCATCGCC CTCG *C*C*C (SEQ ID NO: 17) BFP0/NCA*A*G*TGGTGCGCTCCTGGAC 201-mer GTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCA TGTGGTCGGGGTAGCGGCTGAA GCACTGCACGCCGTGGGTGAAGGTGGTCACGAGGGTGGGCCAGG GCAC GGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGC CG TAGGTGGCATCGCCCTC G *C*C*C (SEQ ID NO: 18)BFP4/C G*G*G*CGAGGGCGATGCCACC 201-mer TACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTC GTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCC CGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCA CCAT *C*T*T (SEQ ID NO: 19) BFP0/CG*G*G*CGAGGGCGATGCCACC 201-mer TACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTC GTGACCACCTTCACCCACGGCGTGCAGTGCTTCAGCCGCTACCC CGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCA CCAT*C*T*T (SEQ ID NO: 20) *= PS linkage(phosphothioate)

Example 2: Conversion Rates Using 5′Cy3/3′idC Labeled GRONs

The purpose of this series of examples is to compare the efficiencies ofphosphothioate (PS) labeled GRONs (having 3 PS moieties at each end ofthe GRON) to the 5′Cy3/3′idC labeled GRONs. The 5′Cy3/3′idC labeledGRONs have a 5′ Cy3 fluorophore (amidite) and a 3′ idC reverse base.Efficiency was assessed using conversion of blue fluorescent protein(BFP) to green fluorescence.

In all three examples, done either by PEG delivery of GRONs intoprotoplasts in individual Falcon tubes (labeled “Tubes”) or in 96-wellplates (labeled “96-well dish”), there was no significant differencebetween the different GRON chemistries in BFP to GFP conversionefficiency as determined by cytometry (FIG. 1).

Example 3: Comparison Between 41-mer BFP4/NC 5′-3PS/3′-3PS GRON and2′-O-Me GRONs

The purpose of this series of examples is to compare the conversionefficiencies of the phosphothioate (PS) labeled GRONs with 3PS moietiesat each end of the GRON to 2′-O-Me or “Okazaki fragment GRONs” in thepresence and absence of a member of the bleomycin family, Zeocin™(1mg/ml) to induce DNA breaks. The designs of these GRONs are depicted inFIG. 2. GRONs were delivered into Arabidopsis thaliana BFP protoplastsby PEG treatment and BFP to GFP conversion was determined at 24 h posttreatment by cytometry. Samples treated with zeocin (1 mg/ml) wereincubated with zeocin for 90 min on ice prior to PEG treatment.

In general the presence of zeocin (1 mg/ml) increased BFP to GFPconversion as determined by cytometry (Table 3). In both the presenceand absence of zeocin, the NC Okazaki GRON containing one 2′-O Me groupon the first RNA base at the 5′ end of the GRON was more efficacious atconverting BFP to GFP when compared to the NC Okazaki GRON containingone 2′-O Me group on each of the first nine 5′ RNA bases (FIG. 2 andTable 3).

In all examples, there was no significant difference between the 41-merBFP4/NC 5′3PS/3′3PS and the 71-mer Okazaki Fragment BFP4/NC GRON thatcontains one 5′ 2′-O me group on the first 5′ RNA base (denoted as BFP471-mer (1) NC) in BFP to GFP conversion in both the presence or absenceof 1 mg/ml of zeocin as determined by cytometry (FIG. 2 and Table 3). Itis important to note that in the presence of zeocin (and expected forbleomycin, phleomycin, tallysomycin, pepleomycin and other members ofthis family of antibiotics) that conversion becomes strand independent(i.e., both coding (C) and non-coding (NC) GRONs with the designs testedin these examples display approximately equal activity).

TABLE 3 Comparison of a standard GRON design with Okazaki fragment GRONdesigns in the presence and absence of a glycopeptide antibiotic zeocin.Exp. BFP4 41-mer BFP4 71-mer (0) BFP4 71-mer (1) BFP4 71-mer (9) Name NCC NC C NC C NC C Zeocin (+) APT043 0.13   0.0875 0.2275 0.2075 0.3550.2275 0.2325 0.195 APT066 1.9    0.713 0.762 0.683 1.318 0.7103 0.7690.883 Mean 1.015   0.40025 0.49475 0.44525 0.8365 0.4689 0.50075 0.539Std Dev 1.251579 0.442295 0.377949 0.336229 0.680944 0.341391 0.3793630.486489 SE 0.885134 0.312797 0.26729 0.237786 0.481573 0.2414360.268291 0.344052 Zeocin (−) APT043 nd nd 0.1875 0.0175 0.21 0.025 0.10.0225 APT066 0.109   0.007 0.112 0.005 0.141 0.023 0.065 0.021 Mean0.109   0.007 0.14975 0.01125 0.1755 0.024 0.0825 0.02175 Std Dev na na0.053387 0.008839 0.04879 0.001414 0.024749 0.001061 SE na na 0.0377560.006251 0.034505 0.001 0.017503 0.00075 BFP4 71-mer (0) 5′ first 10 bpare RNA and GRON has no protection NC C BFP4 71-mer (1) 5′ first 10 bpare RNA and first bp on the 5′ end has a 2′ NC C O—Me BFP4 71-mer (9) 5′first 10 bp are RNA and first nine bp on the 5′ end has a NC C 2′ O—Me

Example 4: Comparison Between 41-mer, 101-mer and 201-mer BFP4/NC5′-3PS/3′-3PS GRONs

The purpose of this series of examples was to compare the conversionefficiencies (in the presence and absence of zeocin) of thephosphothioate (PS) labeled GRONs with 3PS moieties at each end of theGRON of different lengths: 41-mer, 101-mer and 201-mer shown in Table 2.Again, the presence of zeocin (1 mg/ml) increased BFP to GFP conversionrates as determined by cytometry (Table 4). The overall trend in allthree examples was linear with increasing NC GRON length in both thepresence and absence of zeocin. Except for the BFP-4/NC/101 andBFP-4/C/101 in the presence of zeocin, this had conversion rates thatwere close to equal but lower than the 41-mer NC GRON. This is incontrast to all previous examples in which the BFP-4/41 coding andnon-coding GRONs were used, where the non-coding was always far superiorto the coding GRON. This asymmetry in conversion frequency also appliesto the BFP-4/201 GRONs used in this example series.

TABLE 4 Exp. BFP4 41-mer BFP4 101-mer BFP4 201-mer Name NC C NC C NC CZeocin (+) APT038 0.2425 0.1275 0.3025 0.2575 0.97 0.245 APT043 0.130.0875 0.185 0.2275 0.66 0.1875 APT047 0.3975 0.145 0.19 0.125 0.2350.085 APT052 0.3275 nd 0.17 0.21 0.585 0.225 APT052 nd nd 0.3225 0.31750.5075 0.3125 APT058 1.4275 nd 1.2 nd 1.9 nd APT066 1.9 0.713 0.992 1.051.7 0.916 Mean 0.7375 0.26825 0.480286 0.364583 0.936786 0.3285 Std Dev0.7382801 0.297475 0.428968 0.341634 0.630943 0.297412 SE 0.301461860.148738 0.162119 0.139499 0.238452 0.121442 Zeocin (−) APT038 0.05 0.010.1025 0.025 0.5725 0.025 APT066 0.109 0.007 0.214 0.047 0.566 0.035Mean 0.0795 0.0085 0.15825 0.036 0.56925 0.03 Std Dev 0.0417193 0.0021210.078842 0.015556 0.004596 0.007071 SE 0.02950446 0.0015 0.0557580.011002 0.00325 0.005001

Example 5: Delivery of Cas9 Protein into Plants

This example makes use of direct delivery of recombinant Cas9 protein toplant cells as an alternative to delivery of CRISPR-Cas expressionplasmids. This method employs carriers such as cell penetrating peptides(CPP), transfection liposome reagents, poly(ethylene glycol) (PEG)either alone or in combination to allow for delivery of activerecombinant Cas9 protein to cells.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate at 250,000 cellsper well at a cell density of 1×10⁷ cells/ml. Fluorescently-taggedrecombinant Cas9 protein (1 μg) pre-coated with CPPs at 20:1, 10: or 5:1and other CPP to cargo ratio (TAT, Penetratin, Chariot™, PEP-1 or othersfor example) or encapsulated with liposome reagents are then mixed withthe seeded protoplasts and incubated at 23° C. for 2-6 h to allow forCas9/carrier complexes to penetrate the cells. In another series oftreatments fluorescently-tagged recombinant Cas9 protein (1 μg) eitherpre-coated with CPPs as described above or not coated are introduced toprotoplasts using PEG methodology. Protoplasts were then analyzed byflow cytometry 24 h after treatment in order to determine the percentageof Cas9 positive protoplasts within a given treatment.

Example 6: CRISPR with 201-mer±Wobble Base GRONs

The purpose of this series of examples is to demonstrate BFP to GFPconversion in our Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andthe 201-mer GRONs to mediate conversion. The BFP CRISPR targets thecoding strand of the bfp gene and the conversion site is 27 bp upstreamof the PAM sequence (FIG. 3). The GRON is used as a template to repairthe double-stranded break in the bfp gene created by the CRISPR andalong with converting the targeted gene from BFP to GFP, it introduces awobble base in the bfp gene that corresponds to the PAM sequence of theBFP CRISPR as well. A wobble base in the PAM sequence of the BFP CRISPRis hypothesized to minimize re-cutting of the bfp gene by the CRISPRsonce conversion has happened. This series of examples will help toaddress whether or not introducing a wobble base into the PAM sequenceof the BFP CRISPR in converted bfp genes will increase conversionefficiencies.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the mannopine synthase (MAS) promoter driving the Cas9coding sequence with an rbcSE9 terminator and the Arabidopsis U6promoter driving the sgRNA with a poly-T10 terminator (“T10” disclosedas SEQ ID NO:21). The CRISPR plasmids along with GRON were introducedinto protoplasts by PEG mediated delivery at a final concentration of0.05 μg/μl and 0.16 μM respectively. Protoplasts were incubated in thedark at 23° C. for 72 hours, and then they were analyzed by flowcytometer in order to determine the percentage of GFP positiveprotoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. In this example the BFP targets the bfp gene (FIG. 3). 201-merGRONs targeting BFP with or without wobble bases were used to determinetheir effect on the rate of BFP to GFP conversion. Table 5 gives a listof the GRONs and their corresponding sequences.

TABLE 5 List of GRONs used in these examples(SEQ ID NOS: 17, 19, 17 and 22, respectively, in order of appearance)GRON GRON Name Chemistry GRON Sequence BFP4/NC 3PS 5′AAGATGGTGCGCTCCTG201-mer GACGTAGCCTTCGGGCATG GCGGACTTGAAGAAGTCGT GCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGC ACGCCGTAGGTGAAGGTGG TCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTG GTGCAGATGAACTTCAGGG TCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC 3′ BFP4/C 3PS 5′GGGCGAGGGCGATGCCA 201-merCCTACGGCAAGCTGACCCT GAAGTTCATCTGCACCACC GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CTTCACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGA CTTCTTCAAGTCCGCCATG CCCGAAGGCTACGTCCAGGAGCGCACCATCTT 3′ BFP4/NC 3PS 5′AAGATGGTGCGCTCCTG 201-merGACGTAGCCTTCGGGCATG (1 wobble) GCGGACTTGAAGAAGTCGT GCTGCTTCATGTGGTCTGGGTAGCGGCTGAAGCACTGC ACGCCGTAGGTGAAGGTGG TCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTG GTGCAGATGAACTTCAGGG TCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC 3′ BFP4/C 3PS 5′GGGCGAGGGCGATGCCA 201-merCCTACGGCAAGCTGACCCT (1 wobble) GAAGTTCATCTGCACCACC GGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CTTCACCTACGGCGTGCAG TGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGA CTTCTTCAAGTCCGCCATG CCCGAAGGCTACGTCCAGGAGCGCACCATCTT 3′

Results

Using the BFP CRISPR, the BFP4/C GRON with the wobble bases is up to a3.5-fold more efficacious in BFP to GFP conversion when compared to theBFP4/C GRON without the wobble bases (Table 6). There is up to a5.9-fold increase in BFP to GFP conversion when the BFP4/C GRON with thewobble base is used instead of the BFP4/NC GRON with the wobble base(Table 6). Therefore, the BFP4/C GRON with the wobble base is mostefficacious in BFP to GFP conversion when used with the BFP CRISPR.

Conclusions

Including a wobble base in the GRON that changes the PAM sequence of theBFP CRISPR in the converted target gene increases BFP to GFP conversion.BFP to GFP conversion by the BFP CRISPR along with the wobble-based GRONwas confirmed by Next Generation Sequencing (data not shown).Additionally, the ability of the BFP CRISPR to cleave the DNA andproduce indels in the bfp gene was confirmed by Next GenerationSequencing (data not shown).

TABLE 6 The percentage of BFP to GFP conversion as determine bycytometry at 72 h post PEG delivery of the BFP CRISPR and GRON intoprotoplasts derived from the Arabidopsis thaliana BFP transgenic line21-15-09. Percentage of GFP Positive Cells (std dev) CRISPR: BFP5 Exp.BFP4/C GRON BFP4/NC GRON Name (−) Wobbles (+) 1 wobbles (−) Wobbles (+)1 wobbles Exp 1 0.46(0.07) 1.59(0.06) 0.08(0.02) 0.27(0.04) Exp 20.24(0.03) 0.61(0.05) 0.04(0.01) 0.16(0.04)

Example 7: CRISPR with Cy3 Modified GRONs

The purpose of this series of examples is to demonstrate BFP to GFPconversion in our Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andGRONs to mediate conversion. The BFP6 CRISPR (CR:BFP6) used in theseexamples targets the bfp gene and causes a double-stranded break in theDNA near the site of conversion. The GRONs used with the BFP6 CRISPR,contains the coding sequence of the bfp gene around the site ofconversion and are labeled at the 5′ end with Cy3 and at the 3′ end withan idC reverse base and are herein referred to as Cy3 GRONs. These GRONsare tested at three different lengths of 41-mers, 101-mers and 201-mersand they are directly compared to the 3PS GRONs which only differ fromthe Cy3 GRONs in that they have 3 phosphothioate linkages on both the 5′and 3′ ends of the GRON. These GRONs are herein referred to as 3PSGRONs. See Table 7 for the list of GRONs used in these examples.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator (“T10” disclosed as SEQ ID NO:21).The CRISPR plasmids along with GRON were introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR and 8.0 μM for the 41-mer, 0.32 μM for the 101-mer and 0.16 μM201-mer GRONs. GRON treatments alone received a final GRON concentrationafter PEG delivery of 8.0 μM for the 41-mer, 5.0 μM for the 101-mer and2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23° C.for 72 hours, and then they were analyzed by flow cytometer in order todetermine the percentage of GFP positive protoplasts within a giventreatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. In this experiment the BFP6 CRISPR (5′GGTGCCGCACGTCACGAAGTCGG 3′(SEQ ID NO23)) was used which targets the bfp gene. The GRONs containthe coding sequence of the bfp gene near the site of conversion. Table 7gives a list of the GRONs used.

TABLE 7 List of GRONs used in these examples (SEQ ID NOS 24, 15, and 19,respectively, in order of appearance) GRON GRON GRON Name ChemistrySequence CRISPR BFP4/C Cy3 or 5′CCCTCGTGACC BFP6 41-mer 3PSACCTTCACCTACG GCGTGCAGTGCTT CAGC 3′ BFP4/C Cy3 or 5′CCACCGGCAAG BFP6101-mer 3PS CTGCCCGTGCCCT GGCCCACCCTCGT GACCACCTTCACC TACGGCGTGCAGTGCTTCAGCCGCTA CCCCGACCACATG AAGCAGCACGA C 3′ BFP4/C Cy3 or 5′GGGCGAGGGCGBFP6 201-mer 3PS ATGCCACCTACGG CAAGCTGACCCTG AAGTTCATCTGCA CCACCGGCAAGCTGCCCGTGCCCTGG CCCACCCTCGTGA CCACCTTCACCTA CGGCGTGCAGTGC TTCAGCCGCTACCCCGACCACATGAA GCAGCACGACTTC TTCAAGTCCGCCA TGCCCGAAGGCTA CGTCCAGGAGCGCACCATCTT3′

Results

Using the BFP6 CRISPR, the Cy3 GRONs at all lengths tested are able tomediate BFP to GFP conversion as efficiently as the 3PS GRONs (FIG. 4).Overall, the samples containing the BFP6 CRISPR and GRON have higherlevels of BFP to GFP conversion when compared to the GRON only samples(FIG. 4), demonstrating the positive impact CRISPRs have on increasingconversion rates.

Example 8: CRISPR with GRONs of Varying Size

The purpose of this series of examples is to demonstrate BFP to GFPconversion in our Arabidopsis thaliana BFP transgenic model system usingCRISPRs to create targeted double-stranded breaks in the bfp gene andGRONs of varying lengths to mediate conversion. The BFP CRISPR used inthese examples targets the bfp gene and causes a double-stranded breakin the DNA near the site of conversion. The GRONs used with the BFPCRISPR, contains the coding sequence of the bfp gene around the site ofconversion and are labeled at both the 5′ end and the 3′ end with 3phosphothioate linkages and are herein referred to as 3PS GRONs. TheseGRONs are tested at three different lengths of 60-mers, 101-mers and201-mers and they are directly compared to the GRON only treatments. SeeTable 8 for the list of GRONs used in these examples.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21).The CRISPR plasmids along with GRON were introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR and 0.547 μM for the 60-mer, 0.32 μM for the 101-mer and 0.16 μM201-mer GRONs. GRON treatments alone received a final GRON concentrationafter PEG delivery of 7.5 μM for the 60-mer, 5.0 μM for the 101-mer and2.5 μM for the 201-mer. Protoplasts were incubated in the dark at 23° C.for 72 hours, and then they were analyzed by flow cytometry in order todetermine the percentage of GFP positive protoplasts within a giventreatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′GTCGTGCTGCTTCATGTGGT3′ (SEQ IDNO:25). In this example the BFP CRISPR was used which targets the bfpgene. The GRONs contain the coding sequence of the bfp gene near thesite of conversion. Table 8 gives a list of the GRONs used.

TABLE 8 List of GRONs used in these examples.(SEQ ID NOS 26, 27, and 22, respectively, in order of appearance). GRONGRON Name Chemistry GRON Sequence BFP4/C 3PS 5′GTGACCACCTTCA 60-merCCTACGGCGTGCAGT (1 wobble) GCTTCAGCCGCTACC CAGACCACATGAAGC AG 3′ BFP4/C3PS 5′CCACCGGCAAGCT 101-mer GCCCGTGCCCTGGCC (1 wobble) CACCCTCGTGACCACCTTCACCTACGGCGT GCAGTGCTTCAGCCG CTACCCAGACCACAT GAAGCAGCACGAC 3′ BFP4/C3PS 5′GGGCGAGGGCGA 201-mer TGCCACCTACGGCAA (1 wobble) GCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCC CTGGCCCACCCTCGT GACCACCTTCACCTACGGCGTGCAGTGCTT CAGCCGCTACCCAGA CCACATGAAGCAGCA CGACTTCTTCAAGTCCGCCATGCCCGAAGG CTACGTCCAGGA GC GCACCATCTT 3′

Results

Using the BFP CRISPR, GRONs at lengths >101 nt are better at mediatingBFP to GFP conversion when directly compared to the 60 nt long GRONs(FIG. 5). Overall, the samples containing the BFP CRISPR and GRON havehigher levels of BFP to GFP conversion when compared to the GRON onlysamples (FIG. 5), demonstrating the positive impact CRISPRs have onincreasing conversion rates. This data further demonstrates that thelength of the GRON that is most efficacious in mediating BFP to GFPconversion, when used along with the CRISPR, needs to be >101 nt inlength.

Example 9: CRISPR with 2′-O-Me GRONs

The purpose of this examples is to demonstrate BFP to GFP conversion inour Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted double-stranded breaks in the bfp gene and GRONs tomediate conversion. The BFP CRISPR used in this example targets the bfpgene and causes a double-stranded break in the DNA near the site ofconversion. The GRONs used with the BFP CRISPR, contain either thecoding or non-coding sequence of the bfp gene around the site ofconversion with the first ten 5′ bases of the GRON being RNA basesinstead of DNA bases. These RNA bases are labeled with 2′-O-Me group(s)at either the first 5′ RNA base or the first nine 5′ RNA bases asdepicted in FIG. 6. These GRONs are herein referred to as 2′-O-Me GRONsand are directly compared to the 3PS GRONs of similar lengths thatcontain DNA bases with 3 phosphothioate linkages on both the 5′ and 3′ends of the GRON. These GRONs are herein referred to as 3PS GRONs. SeeTable 9 for the list of GRONs used in these examples.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and the Arabidopsis U6 promoter driving the sgRNAwith a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21). The sgRNAis a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).The CRISPR plasmids along with GRON were introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR, 0.5 μM for the 71-mer and 0.16 μM for the 201-mer GRONs. GRONtreatments alone received a final GRON concentration after PEG deliveryof 5.0 μM for the 71-mer and 2.5 μM for the 201-mer. Protoplasts wereincubated in the dark at 23° C. for 72 hours, and then they wereanalyzed by flow cytometer in order to determine the percentage of GFPpositive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′CTCGTGACCACCTTCACCCA 3′ (SEQID NO:28). In this example the BFP CRISPR was used which targets the bfpgene. The GRONs contain either the coding or non-coding sequence of thebfp gene near the site of conversion. Table 9 shows a list of the GRONsused.

TABLE 9 List of GRONs used in these examples(SEQ ID NOS 29,30, 19,17, 29, 31,19, and 32,respectively, in order of appearance) GRON Name GRON ChemistryGRON Sequence BFP4/C 3PS 5′GCUGCCCGUGCCCTGGCC 71-merCACCCTCGTGACCACCTTCA CCTACGGCGTGCAGTGCTTC AGCCGCTACCCCG3′ BFP4/NC 3PS5′TTCATGTGGTCGGGGTAG 71-mer CGGCTGAAGCACTGCACGCC GTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGG3′ BFP4/C 3PS 5′GGGCGAGGGCGATGCCAC 201-merCTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGC AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCA CCTACGGCGTGCAGTGCTTC AGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGC TACGTCCAGGAG CGCACCATCTT3′BFP4/NC 3P 5′AAGATGGTGCGCTCCTGG 201-mer ACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCT GCTTCATGTGGTCTGGGTAG CGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGA GGGTGGGCCAGGGCACGGGC AGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGC CGTAGGTGGCATCGCCCTCG CCC3′ BFP4/C 2′-O-Me5′gcugcccgugCCCTGGCC 71-mer CACCCTCGTGACCACCTTCA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG 3′ BFP4/NC 5′uucaugugguCGGGGTAG 71-merCGGCTGAAGCACTGCACGCC GTAGGTGAAGGTGGTCACGA GGGTGGGCCAGGG3′ BFP4/C 2′-O-Me5′gggcgagggcGATGCCAC 201-mer CTACGGCAAGCTGACCCTGA AGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC CACCCTCGTGACCACCTTCA CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACAT GAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT CTT3′ BFP4/NC 2′-O-Me 5′aagauggugcGGTCCTGG 201-merACGTAGCCTTCGGGCATGGC GGACTTGAAGAAGTCGTGCT GCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCC GTAGGTGAAGGTGGTCACGA GGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGAT GAACTTCAGGGTCAGCTTGC CGTAGGTGGCATCGCCCTCG CCC3′

Results

The 71-mer and 201-mer 2′-O-Me GRONs had similar BFP to GFP conversionwhen compared to the various different types of GRON protections of (0),(1) or (9) using the BFP CRISPRs (FIGS. 7 and 8). The 2′-O-Me GRONs aremore efficacious than their 3PS GRON counterparts at mediating BFP toGFP conversion using the BFP CRISPRs (FIGS. 7 and 8).

Example 10: CRISPR Nickases with GRONs Introduction

The purpose of this examples is to demonstrate BFP to GFP conversion inour Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted single-stranded nicks in the bfp gene and GRONs tomediate conversion. The BFP1 CRISPR (CR:BFP1) used in this exampletargets the bfp gene and contains mutations in the catalytic residues(D10A in RuvC and H840A in HNH) that causes single-stranded nicks in theDNA of the bfp gene near the site of conversion on either the DNAstrands complementary or non-complementary to the guide RNArespectively. These CRISPRs are herein referred to as BFP1 CRISPRnickase D10A and BFP1 CRISPR nickase H840A and are used either alone orwith BFP5 sgRNA on a separate plasmid. When multiple CRISPR nickases areused together in this example, they can either nick the same DNA strandor opposite DNA strands. When both Cas9 proteins that contain the samemutations, either D10A or H840A, are used together, they nick the samestrand of DNA. Conversely, when two Cas9 proteins are used together andone of them contains the D10A mutation and the other one contains theH840A mutation, they nick opposite strands of the DNA. The GRONs usedwith the nickase CRISPRs, contains either the coding or the non-codingsequence of the bfp gene around the site of conversion with one wobblebase located in the PAM sequence of BFP5 CRISPR. These GRONs have 3phosphothioate linkages on both the 5′ and 3′ ends and are hereinreferred to as 3PS GRONs. See Table 10 for the list of GRONs used inthese examples. The nickase CRISPRs are directly compared to theirCRISPR counterparts that are able to cause targeted double-strandedbreaks in the DNA of the bfp gene.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence withan rbcSE9 terminator and the Arabidopsis thaliana U6 promoter drivingthe sgRNA with a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21).The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activating crRNA(tracrRNA). The Cas9 gene contains mutations in the catalytic residues,either D10A in RuvC or H840A in HNH. The CRISPR plasmids along with GRONare introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl for the CRISPR and 0.16 μM for the 201-mer.GRON treatments alone received a final GRON concentration after PEGdelivery of 2.5 μM for the 201-mer. Protoplasts were incubated in thedark at 23° C. for 72 hours, and then they were analyzed by flowcytometer in order to determine the percentage of GFP positiveprotoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. In this example the BFP1 and BFP5 sgRNA was used that targetsdifferent regions the bfp gene near the site of conversion. The BFP1spacer (5′CTCGTGACCACCTTCACCCA 3′(SEQ ID NO:28)) targets thecoding-strand while the BFP5 spacer (5′GTCGTGCTGCTTCATGTGGT3′ (SEQ IDNO:25)) targets the non-coding strand of the bfp gene. The GRONs containeither the coding or non-coding sequence of the bfp gene near the siteof conversion. Table 10 shows a list of the GRONs used.

TABLE 10 List of GRONs used in these examples (SEQ ID NOS 17 and 22,respectively, in order of appearance) GRON GRON GRON Name ChemistrySequence BFP4/NC 3PS 5′ AAGATGGTGCGCT 201-mer CCTGGACGTAGCCTTC(1 wobble; GGGCATGGCGGACTTG BFP5) AAGAAGTCGTGCTGCT TCATGTGGTCTGGGTAGCGGCTGAAGCACTGC ACGCCGTAGGTGAAGG TGGTCACGAGGGTGGG CCAGGGCACGGGCAGCTTGCCGGTGGTGCAGA TGAACTTCAGGGTCAG CTTGCCGTAGGTGGCA TCGCCCTCGCCC 3′BFP4/C 3PS 5′ GGGCGAGGGCGAT 201-mer GCCACCTACGGCAAGC (1 wobble;TGACCCTGAAGTTCAT BFP5) CTGCACCACCGGCAAG CTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CTTCACCTACGGCGTG CAGTGCTTCAGCCGCT ACCCAGACCACATGAAGCAGCACGACTTCTTC AAGTCCGCCATGCCCG AAGGCTACGTCCAGGA GCGCACCATCTT 3′

Results

Both of the CRISPR nickases (D10A and H840A) are more efficient atmediating BFP to GFP conversion when the BFP1 CRISPR and the BFP5 CRISPRwere used together instead of separately (FIG. 9). In addition, when theBFP1 and BFP5 D10A CRISPR nickases are used together with the C/201 1WGRON, the BFP to GFP conversion is significantly higher when compared totreatments where these CRISPR nickases are used with the NC/201 1W GRON(FIG. 9). When the BFP1 and BFP5 H840A CRISPR nickases are used togetherroughly the same level of BFP to GFP conversion is observed with eitherthe C/201 or NC/201 1W GRONs (FIG. 9). These levels of BFP to GFPconversion are slightly higher than when the BFP5 CRISPR is used aloneand slightly lower than when the BFP1 CRISPR is used alone (FIG. 9).

Example 11: Use of CRISPRs to Target Multiple Genes

The purpose of this example is to demonstrate conversion of multiplegenes simultaneously in a given population of protoplasts derived fromthe Arabidopsis thaliana model system using CRISPRs to createdouble-stranded breaks in targeted genes and GRONs to mediateconversion. The CRISPRs used in this example target both the BFP andacetohydroxy acid synthase (AHAS) genes in the Arabidopsis thalianagenome by introducing into protoplasts plasmid(s) encoding the Cas9 geneand multiple sgRNA targeting these two different genes. The sgRNA is afusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Thiswill allow Cas9 to cause double-stranded breaks in both the BFP and AHASgenes in the presence of GRONs that will mediate their conversion.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×107 cells/ml. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis thaliana U6 promoter driving multiple different sgRNAswith a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21). The sgRNAis a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).The CRISPR plasmids along with GRON are introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR and 0.16 μM for the 201-mer. GRON treatments alone receive afinal GRON concentration after PEG delivery of 2.5 μM for the 201-mer.Protoplasts will be incubated in the dark at 23° C. for 72 hours, andthen they are analyzed by flow cytometer and an allele specific PCRassay in order to determine the percentage of both BFP to GFP and AHASconverted protoplasts respectively within a given treatment.

In the an allele specific PCR assay 10-16 replicates of 5,000 genomeequivalents of genomic DNA were used in the primary PCR reactions.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In this example different sgRNAs and GRONs are usedto target multiple genes near their sites of conversion; BFP spacer(5′CTCGTGACCACCTTCACCCA 3′ (SEQ ID NO:28)) and AHAS spacer (5′TGGTTATGCAATTGGAAGATCGC 3′(SEQ ID NO:33). Table 11 describes the GRONsused.

TABLE 11 List of GRONs used in this example(SEQ ID NOS 19 and 34, respectively, in order of appearance)  GRONTarget GRON Name Gene Chemistry GRON Sequence BFP/C BFP 3PS5′GGGCGAGGGCGATGCCACCTAC 201-mer GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG CCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTC AGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC ATGCCCGAAGGCTACGTCCAGGAG CGCACCATCTT3' AHAS(W)AHAS 3PS 5′AGCTGCTGCAAACAGCAACATG 574/NC TTCGGGAATATCTCGTCCTCCTGA201-mer GCCGGATCCCCGAGAAATGTGTGA GCTCGGTTAGCTTTGTAGAAGCGATCTTCCAATTGCATAACCATGCCA AGATGCTGGTTGTTTAATAAAAGTACCTTCACTGGAAGATTCTCTACA CGAATAGTGGCTAGCTCTTGCACA TTCATTATAAA3'

Results

BFP to GFP and AHAS conversion was determined at 144 h post PEG deliveryof the BFP and AHAS CRISPR plasmids and the BFP/C 201-mer andAHAS(W)574/NC 201-mer GRONs into the Arabidopsis thaliana BFP transgenicline. Flow cytometry data revealed that Treatment 1 resulted in 0.20%BFP to GFP conversion (Table 12). Allele specific PCR assay revealedthat Treatment 1 resulted in 0.01% AHAS converted protoplasts (Table12). GRON only treatments had minimal conversion using both assays(Table 12). This example demonstrates the successful simultaneousconversion of two independent target genes (BFP and AHAS) within a givenpopulation of protoplasts derived from the Arabidopsis thaliana BFPmodel system.

TABLE 12 Measurement of conversion of both the BFP and AHAS genes at 144h post PEG delivery of CRISPR plasmids and GRONs into a given populationof protoplasts derived from the Arabidopsis thaliana BFP model systemeither by: (1) flow cytometry which determines the percentage of GFPpositive protoplasts or (2) allele specific PCR which determines thepercentage of AHAS converted protoplasts. BFP to AHAS- GFP W574Lconversion Conversion Treat- Flow Allele ment CRISPR GRONs CytometrySpecific PCR 1 CR-BFP and BFP/C 201-mer and 0.20%  ~0.01% CR-AHASAHAS(W)574/ NC 201-mer 2 None BFP/C 201-mer and 0.01% ~0.001%AHAS(W)574/ NC 201-mer 3 None None 0.01% ~0.001%

Example 12: Delivery of Cas9 mRNA into Plant Cells

This example makes use of direct delivery of recombinant Cas9 mRNA intoplant cells as an alternative to delivery of CRISPR-Cas expressionplasmids. This method includes (1) in vitro synthesis of modified mRNAand (2) Delivery of this modified mRNA into plant cells.

Methods

A Cas9 mRNA will be transcribed in vitro using an RNA polymerase such asT7, T3 or SP6 from a linearized plasmid template including components ofa 5′UTR, the coding sequence (CDS) for the protein and a 3′UTR. One RNApolymerase may incorporate a particular modified nucleoside better thananother. The 5′ UTR may contain elements that improve its stability suchas the MiR-122 of hepatitis C virus (Shimakami et al., 2012). In vitrosynthesis will incorporate nucleosides that are protective and ensuregood translation in the target plant cells. Recombinant Cas9 mRNA willbe capped and contain a polyA tail.

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate at 250,000 cellsper well at a cell density of 1×107 cells/ml. Recombinant Cas9 mRNA willbe delivered into plant cells in one of the following means (list notinclusive): cell penetrating peptides (CPP), transfection liposomereagents, poly(ethylene glycol) (PEG) either alone or in combination toallow for delivery of active recombinant Cas9 mRNA into cells.

Example 13: CRISPR-Cas for Tethering DNA, RNA or Proteins

This example makes use of a modified single guide RNA (sgRNA) cassettewherein a linker sequence (may also be referred to as a tetheringsequence) is included at the 3′ end of the tracrRNA but 5′of the RNApolymerase III termination signal as shown in the example below (FIG.10). The sgRNA is a fusion of CRISPR RNA (crRNA) and trans-activatingcrRNA (tracrRNA). Though preferred, the placement of the linker is notlimited to the 3′ end of the tracerRNA but will be investigated atseveral positions within the sgRNA cassette. The linker sequence mayvary in nucleotide length or contain secondary structure that wouldimprove tethering or increase the number of molecules tethered throughtriplex interactions.

The linker will allow for Watson-Crick base pairing with a DNA, RNA orproteins that contains the complementary sequence (FIG. 10).Additionally, linker sequence in sgRNA cassettes will be designed tocontain advanced secondary and tertiary structure allowing for morecomplex multifaceted interaction regions that would tether multiple DNA,RNA or protein molecules.

The overall concept is that a CRISPR-Cas complex will tether biologicalmolecules to the site of nuclease activity thereby increasing thelikelihood of gene editing. These biological molecules include the GRONwhich will mediate conversion of targeted gene(s). Tethering linkers canbe added to sgRNA by simply using, for example, Gene Strings or annealedoligos.

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue are seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR-Cas tetheringplasmids contains the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and

Arabidopsis thaliana U6 promoter driving sgRNAs with a linker sequencethat is complementary to a polynucleotide tract of 15-30 bp located on a201-mer editing GRON targeting bfp (as shown in FIG. 10). The sgRNAtethering cassette is terminated by a poly-T10 terminator (“T10”disclosed as SEQ ID NO: 21). The CRISPR-Cas plasmids along with GRON areintroduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl for the CRISPR and 0.16 μM of the 201-merGRON. GRON treatments alone received a final GRON concentration afterPEG delivery of 2.5 μM for the 201-mer. Protoplasts were incubated inthe dark at 23° C. for 72 hours, and then they were analyzed by flowcytometer in order to determine the percentage of GFP positiveprotoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which areexpressed from the same or different plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) and linker. ThecrRNA region contains the spacer sequence used to guide the Cas9nuclease to the BFP target gene. In this example CRISPR is used whichtargets the bfp gene. The GRONs contain either the coding or non-codingsequence of the bfp gene near the site of conversion.

Example 14: CRISPRs with Truncated gRNA

The purpose of this example is to demonstrate conversion of BFP to GFPin protoplasts derived from the Arabidopsis thaliana BFP model systemusing CRISPRs to create double-stranded breaks in targeted genes andGRONs to mediate conversion. The CRISPRs used in this example targetsthe bfp gene in the Arabidopsis thaliana genome by introducing intoprotoplasts plasmid(s) encoding the Cas9 gene and one sgRNAs that is twodifferent lengths. The sgRNA is a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA). The crRNA which guides the Cas9 tothe target genes is called the spacer and it is typically 20-nt inlength (CR:BFP1 20-nt), however, in these examples we tested theeffectiveness of using a smaller length spacer of 17-nt (CR:BFP1 17-nt)in mediating BFP to GFP conversion.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×107 cells/ml. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis thaliana U6 promoter driving multiple different sgRNAswith a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21). The sgRNAis a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).The CRISPR plasmids along with GRON are introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR and 0.16 μM for the 201-mer. GRON treatments alone receive afinal GRON concentration after PEG delivery of 2.5 μM for the 201-mer.Protoplasts will be incubated in the dark at 23° C. for 72 hours, andthen they are analyzed by flow cytometer in order to determine thepercentage of BFP to GFP within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In these examples, two different length BFP1 spacersof 20-nt (5′CTCGTGACCACCTTCACCCA 3′ (SEQ ID NO:28)) vs. 17-nt(5′GTGACCACCTTCACCCA 3′(SEQ ID NO:35)) were tested. Table 13 describesthe GRON used

TABLE 13 List of GRON used in this example (SEQ ID NO: 36) GRON GRONName Chemistry GRON Sequence BFP4/NC 3PS 5′AAGATGGTGCGCTCCTGGACGTAGCCTTC201-mer GGGCATGGCGGACTTGAAGAAGTCGTGCTGC 3WTTCATGTGGTCGGGGTAGCGGCTGAAGCACT GCACGCCGTACGTAAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTG CAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC3′

Results

Reducing the length of the BFP1 protospacer from 20bp to 17bp hadsimilar levels of BFP to GFP conversion of 0.163% vs. 0.177%respectively at 72 h post PEG delivery of plasmids and GRONs into theArabidopsis thaliana BFP model system (FIG. 11).

Example 15: CRISPRs with Amplicon gRNA

The purpose of this Example is to demonstrate conversion of BFP to GFPin protoplasts derived from the Arabidopsis thaliana BFP model systemusing CRISPRs to create double-stranded breaks in targeted genes andGRONs to mediate conversion. The CRISPRs used in this Example targetsthe bfp gene in the Arabidopsis thaliana genome by introducing intoprotoplasts plasmid(s) encoding the Cas9 gene and one sgRNAs that iseither encoded on a plasmid or introduced into protoplasts as anamplicon. The sgRNA is a fusion of CRISPR RNA (crRNA) andtrans-activating crRNA (tracrRNA). The crRNA guides the Cas9 to thetarget genes, where Cas9 creates a double-stranded break and the GRON isused as a template to convert BFP to GFP in a site-directed manner.

Methods

Arabidopsis thaliana protoplasts derived from induced root tissue areseeded on a flat-bottom 96-well plate, at 250,000 cells per well at acell density of 1×10⁷ cells/ml. The CRISPR encoded plasmids contain theMAS promoter driving the Cas9 coding sequence with a rbcSE9 terminatorand Arabidopsis U6 promoter driving multiple different sgRNAs with apoly-T₁₀ terminator (“T10” disclosed as SEQ ID NO: 21). The sgRNA is afusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). TheCRISPR plasmids along with GRON are introduced into protoplasts by PEGmediated delivery at a final concentration of 0.05 μg/μl for the CRISPRand 0.16 μM for the 201-mer. GRON treatments alone receive a final GRONconcentration after PEG delivery of 2.5 μM for the 201-mer. Protoplastswill be incubated in the dark at 23° C. for 72 hours, and then they areanalyzed by flow cytometer in order to determine the percentage of BFPto GFP within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same or multiple plasmids. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer sequence used to guide the Cas9 nuclease tothe targeted genes. In these examples, the same BFP6 gRNA(5′GGTGCCGCACGTCACGAAGTCGG 3′ (SEQ ID NO:23)) was delivered intoprotoplasts either as an amplicon or encoded on a plasmid. Table 14describes the GRONs used.

TABLE 14 List of GRONs used in this example(SEQ ID NOS 17 and 19, respectively, in order of appearance)  GRON GRONName Chemistry GRON Sequence BFP4/NC 3PS 5′AAGATGGTGCGCTCCTGGACGTAGCCTTC201-mer GGGCATGGCGGACTTGAAGAAGTCGTGCTGC TTCATGTGGTCTGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGT GGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGG TGGCATCGCCCTCGCCC3′ BFP4/C 3PS5′GGGCGAGGGCGATGCCACCTACGGCAAGC 201-mer TGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACC ACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTT CTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT3′

Results

Delivery of the BFP6 gRNA as an amplicon (CR:BFP6 (gRNA amplicon)) alongwith a plasmid containing only Cas9 had similar rates of BFP to GFPconversion when compared to treatments with both the gRNA (gRNA plasmid)and Cas9 being encoded on separate plasmids at 72 h post PEG delivery ofplasmids and GRONs into the Arabidopsis thaliana BFP model system (FIG.12).

Example 16: CRISPRs with Unmodified GRONs

The purpose of this example is to demonstrate BFP to GFP conversion inour Arabidopsis thaliana BFP transgenic model system using CRISPRs tocreate targeted double-stranded breaks in the bfp gene and GRONs tomediate conversion. The BFP CRISPR used in this example targets the bfpgene and causes a double-stranded break in the DNA near the site ofconversion. The 3PS GRONs contain DNA bases with 3 phosphothioatelinkages on both the 5′ and 3′ ends of the GRON and are herein referredto as 3PS GRONs. The 3PS GRONs were directly compared to theirunmodified GRON counterparts in mediated BFP to GFP conversion using theBFP CRISPRs in our BFP transgenic Arabidopsis thaliana model system. SeeTable 15 for the list of GRONs used in these examples

TABLE 15 List of GRONs used in this example(SEQ ID NOS 24 and 37, respectively,  in order of appearance) GRON GRONName Chemistry GRON Sequence BFP4/C 3PS 5′CCCTCGTGACCACCTTCACCTACGGCGTG41-mer CAGTGCTTCAGC3′ BFP4/NC None 5′GCTGAAGCACTGCACGCCGTAGGTGAAGG41-mer TGGTCACGAGGG3′

Methods

BFP transgenic Arabidopsis thaliana protoplasts derived from inducedroot tissue were seeded on a flat-bottom 96-well plate, at 250,000 cellsper well at a cell density of 1×107 cells/ml. The CRISPR encodedplasmids contain the MAS promoter driving the Cas9 coding sequence witha rbcSE9 terminator and the Arabidopsis U6 promoter driving the sgRNAwith a poly-T10 terminator (“T10” disclosed as SEQ ID NO: 21). The sgRNAis a fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).The CRISPR plasmids along with GRON were introduced into protoplasts byPEG mediated delivery at a final concentration of 0.05 μg/μl for theCRISPR, 0.16 μM for the 41-mer GRONs. GRON treatments alone received afinal GRON concentration after PEG delivery of 0.8 μM for the 41-mer.Protoplasts were incubated in the dark at 23° C. for 72 hours, and thenthey were analyzed by flow cytometer in order to determine thepercentage of GFP positive protoplasts within a given treatment.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the BFP targetgene. The BFP CRISPR spacer sequence is 5′CTCGTGACCACCTTCACCCA 3′ (SEQID NO:28). In this example the BFP CRISPR was used which targets the bfpgene. The GRONs contain the non-coding sequence of the bfp gene near thesite of conversion. Table 16 shows a list of the GRONs used.

TABLE 16 List of GRONs used in this example.(SEQ ID NOS 24 and 37, respectively,  in order of appearance). GRON GRONName Chemistry GRON Sequence BFP4/C 3PS 5′CCCTCGTGACCACCTTCACCTACGGCGTG41-mer CAGTGCTTCAGC3′ BFP4/NC None 5′GCTGAAGCACTGCACGCCGTAGGTGAAGG41-mer TGGTCACGAGGG3′

Results

The 41-mer 3PS GRONs are more efficacious than their unmodified GRONcounterparts at mediating BFP to GFP conversion using the BFP CRISPRs(FIG. 13).

Example 17: TALENs and GRONs in Flax

The purpose of this example is to demonstrate EPSPS conversion in flaxat both 24 hours in protoplasts and 3-weeks in microcalli after deliveryof TALEN plasmids and GRONs. The TALENs used in this example targets theepsps gene in the Linum usitatissimum genome by introducing into shoottip derived protoplasts plasmid(s) encoding TALENs creates adouble-stranded break and the GRON is used as a template to convert theepsps gene in a site-directed manner.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The TALEN plasmids along with GRONs wereintroduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl and 0.5 μM respectively. Protoplasts wereincubated in the dark at 25° C. for up to 48 h in liquid medium, orembedded in alginate beads (5×10⁵ cells/ml), and cultured in liquidmedium to induce cell division and the formation of microcalli.Protoplasts or microcalli samples obtained 24 h or 3 weeks after DNAdelivery were analyzed by NGS to determine the percentage of cells (DNAreads) carrying out the target mutation within a given treatment. Thepercent of indels generated by imperfect NHEJ-mediated DNA repair wasalso estimated.

TALEN constructs include two arms (left and right), each consisting of aTAL effector-like DNA binding domain linked to a catalytic DNA cleavagedomain of Fold. The TAL effector-like DNA binding domain guides theTALEN arms to specific sites of DNA which allows the Fold endonucleasesof each arm to dimerize together and cleave double,-stranded DNA. TheTALEN encoded plasmids contains MasP::LuEPSPS_(Leftarm)-T2A-LuEPSPS_(right arm) with a rbcSE9 terminator. LuEPSPS_(leftarm) sequence is 5′TGGAACAGCTATGCGTCCG 3′ (SEQ ID NO:38) and theLuEPSPS_(right arm) sequence is 5′TGAGTTGCCTCCAGCGGCT 3′ (SEQ ID NO:39).GRONs (144-mers) targeting LuEPSPS with or without wobble bases wereused to determine their effect on rate of conversion.

Results

24 hour protoplasts and 3-week old microcalli have 0.067% and 0.051%EPSPS conversion respectively as determined by Next GenerationSequencing (FIG. 14). Additionally, these data show that the TALEN isactive and able to cleave the epsps target gene in Linum usitatissimumand form indels of 2.60% and 1.84% respectively at 24 hours inprotoplasts and up to 3-week in microcalli. Moreover, EPSPS conversionand indels are maintained up to 3 weeks after the TALEN plasmid and GRONare introduced.

Example 18: CRISPRs and GRONs in Flax

The purpose of this example is to demonstrate activity of Cas9 in flaxmicrocalli three and six weeks after delivery of a Cas9 plasmid. TheCRISPRs used in this example targets the epsps gene in the Linumusitatissimum genome by introducing into shoot tip derived protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target genes, where Cas9 creates adouble-stranded break in the epsps gene in a site-directed manner. Thedouble-stranded breaks in the epsps gene when repaired by the ubiquitousNHEJ pathway will cause indels to form around the cleavage site.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The CRISPR encoded plasmids contains the MASpromoter driving the Cas9 coding sequence with an rbcSE9 terminator andthe Arabidopsis thaliana U6 promoter driving the sgRNA with a poly-Tioterminator (“T10” disclosed as SEQ ID NO: 21). The CRISPR plasmids wereintroduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl. Protoplasts were embedded in alginate beads(5×10⁵ cells/ml), cultured in liquid medium, and incubated in a rotatoryshaker (30 rpm) in the dark at 25° C. Microcalli developed fromindividual cells were analyzed by NGS, 3 and 6 weeks after CRISPRplasmid delivery, to determine the percentage of cells (DNA reads)carrying out indels generated by the error-prone NHEJ-mediated DNArepair pathway.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence used to guide the Cas9 nuclease to the target gene.In this example the CRISPR targets the epsps gene.

Results

3- and 6-week old microcalli have 46.5% and 54.7% indel formationrespectively as determined by Next Generation Sequencing (FIG. 15).These data shows that Cas9 is active and able to cleave the EPSPS targetgene in Linum usitatissimum and form indels. Moreover, these indels aremaintained up to 6 weeks after the CRISPR plasmid was introduced.

Example 19: Construction of Engineered Nucleases

CRISPR-Cas

For construction of transient CRISPR-Cas9 expression plasmids, a higherplant codon-optimized SpCas9 gene containing a SV40 NLS at both the N-and C-terminal and a 2× FLAG tag on the N-terminal was synthesized as aseries of GeneArt® Strings™ (Life Technology, Carlsbad, Calif.), thencloned downstream of the mannopine synthase (MAS) promoter and upstreamof the pea ribulose bisphosphate carboxylase (rbcsE9) terminator byGibson's method. Next, an sgRNA cassette consisting of a chimeric gRNAwhose expression is driven by the Arabidopsis U6 promoter, wassynthesized as GeneArt® Strings™, then shuttled into the Cas9 containingconstruct using Gibson's method forming pBCRISPR. To specify thechimeric gRNA for the respective target sequence, pairs of DNAoligonucleotides encoding the variable 20-nt sgRNA targeting sequences(Extended Data Table 2) were annealed to generate short double strandfragments with 4-bp overhangs. The fragments were ligated into BbsIdigested pBCrispr to yield CRISPR-Cas constructs BC-1, BC-2 and BC-3.

TALEN

Design and construction of TALEN expression constructs BT-1 and LuET-1was based on rules as described in Cermak et al., Nucleic Acids Res. 39,e82 (2011). The target sequence was selected based on the gene editingsite and the repeat variable i-residue (RVD) following the rules thatNG, HD, NI, and NN recognize T, C, A, and G, respectively. The assemblyof TAL effector domain linked to the heterodimeric FokI domains wascompleted through a commercial service (GeneArt; Life Technologies).TALEN monomers were cloned downstream of the MAS promoter and upstreamof the rbcE9 terminator using Gibson's method and expressed as a 2Acoupled unit.

Cell Culture and Protoplast Isolation

Surface-sterilized Arabidopsis seeds were germinated on solid ½ MSmedium (Minerals and vitamins according to XX; ½ concentrated; 87.7 mMsucrose) at 28° C. under a 12 h light/dark cycle. Root material from 2to 3-week-old seedlings were collected and maintained in ½ MS liquidmedium under low light conditions at 28° C. Root cultures weretransferred and maintained in MSAR[0.22% ½ MS, 87.7 mM sucrose, 11.4 μMIAA, 2.3 μM 2,4-D, 1.5 μM 2iP, pH 5.8] three weeks prior to protoplastisolation. Roots were cut into approximately 6 mm segments and incubatedin MSAP solution[0.22% 1/2 MS, 87.7 mM sucrose, 11.4 μM IAA, 2.3 μM2,4-D, 1.5 μM 2 iP, and 400 mM mannitol, pH 5.8] containing cell walldigesting enzymes [1.25% Cellulase RS, 0.25% Macerozyme R-10, 0.6 Mmannitol, 5 mM MES, 0.1% BSA] for 3-4 h in the dark with gentle shaking.The released protoplasts were collected and passed through a sterile 100p.m filter and 35 p.m filter. The protoplast filtrate was mixed with 0.8times the volume of Optiprep™ Density Gradient Medium (Sigma) and mixedgently. A 60% W5 [154 mM NaCl, 5 mM KCl, 125 mM CaCl₂.2H₂O, 5 mMglucose, 10 mM MES, (pH 5.8)]/40% Optiprep solution followed by 90%W5/10% Optiprep solution were slowly layered onto the filtrate/Optiprepsolution to make a gradient, which was centrifuged at 198 RCF for 10min. The white protoplast layer was collected and mixed with 2 times thevolume of W5. Protoplasts were centrifuged at 44 RCF for 10 min andre-suspended in TM solution [14.8 mM MgCl₂.6H₂O, 5 mM MES, 572 mMmannitol, (pH 5.8)] at a density of 1×10⁷cells/ml. For experiments withZeocin™ (Life Technologies, Carlsbad, Calif.) and phleomycin (InvivoGen,San Diego, Calif.), protoplasts were kept in TM adjusted to pH 7.0 for90 min on ice before transfection. For antibiotic concentrations seeExtended Data FIG. 1.

Flax protoplasts were isolated from shoot tips obtained from 3-week-oldseedlings germinated in vitro. Shoot tips were finely chopped with ascalpel, pre-plasmolyzed for 1 h at room temperature in B-medium [B5salts and vitamins (Gamborg et al., 1968), 4 mM CaCl₂, 0.1 M glucose,0.3 M mannitol, 0.1 M glycine, 250 mg/l casein hydrolysate, 10 mg/lL-cystein-HCL, 0.5% polyvinylpyrrolidone (MW 10,000), 0.1% BSA, 1 mg/lBAP, 0.2 mg/l NAA, and 0.5 mg/l 2,4-D], and incubated in a cell walldigesting enzyme solution containing B-medium supplemented with 0.66%Cellulase YC and 0.16% Macerozyme R-10 over a rotatory shaker (50 rpm)at 25 ° C. for 5 h. Released protoplasts were sieved and purified bydensity gradient centrifugation using Optiprep (Sigma) layers, countedwith a hemocytometer, and kept stationary overnight in the dark at adensity of 0.5×10⁶ protoplasts/ml in B medium.

Protoplast Transfection

In a 96-well flat bottom plate, 2.5×10⁵ cells per well were transfectedwith 10 pmol GRON, 10 pmol GRON plus 3.25 μg CRISPR-Cas or TALENexpression construct or mock using PEG [270 mM mannitol, 67.5 mMCa(NO3)₂, 38.4% PEG 1500, (pH 5.8)]. Cells and DNA were incubated withPEG on ice for 30 minutes followed by a wash with 200 μl of W5 solution.Finally, 85 μl of MSAP++ [MSAP containing 50 nM phytosulfokine-α and 20μM n-propyl gallate] was added and the cells cultured in low lightconditions at 28° C.

After about 18 h of culture, protoplasts were transfected with TALENplasmid along with GRONs (20 μg plasmid and 0.2 nmol GRON/10⁶protoplasts) using PEG mediated delivery. Treated protoplasts wereincubated in the dark at 25° C. for up to 48 h in B medium, or embeddedin alginate beads 24 h after transfection, and cultured in V-KM liquidmedium to induce cell division and the formation of microcalli. For theantibiotic experiments, 1.25×10⁵ cells per well were transfected with 8μM GRON CG13 using the PEG solution described above.

Cytometry

Seventy-two h after transfection, cells were analyzed by cytometry usingthe Attune® Acoustic Focusing cytometer (Applied Biosystems®) withexcitation and detection of emission as appropriate for GFP. Backgroundlevel was based on PEG-treated protoplasts without DNA delivery. Forantibiotic experiments, protoplasts treated with Zeocin or phleomycinprior to transfection were analyzed by cytometry 48 h aftertransfection.

Sequencing Analysis

Genomic DNA was extracted from CRISPR-Cas or TALEN-treated protoplastsusing the NucleoSpin® Plant II kit as per the manufacturer'srecommendations (Machery-Nagel, Bethlehem, Pa.). Amplicons flanking theTALEN or CRISPR target region were generated using Phusion® polymerasewith 100 ng of genomic DNA and primers BFPF-1(5′-GGACGACGGCAACTACAAGACC-3′ (SEQ ID NO:40))/BFPR-1(5′-TAAACGGCCACAAGTTCAGC-3′(SEQ ID NO:41)) for Arabidopsis CRISPR andTALEN; or LuEPF-1 (5′-GCATAGCAGTGAGCAGAAGC-3′ (SEQ ID NO:42))/LuEPR-15′-AGAAGCTGAAAGGCTGGAAG-3′ (SEQ ID NO:43) for L. usitatissirnurn TALEN.The amplicons were purified and concentrated using Qiaquick MinElutecolumns (Qiagen, Valencia, Calif.). Deep sequencing of the amplicons wasperformed by GeneWiz (South Plainfield, N.J.) using a 2×250 bp MiSeq run(Illumina, San Diego, Calif.). For data analysis fastq files for readland read 2 were imported into CLC Genomics Workbench 7.0.4 (CLCBio,Boston, Mass.). Paired reads were merged into a single sequence if theirsequences overlapped. A sequence for an amplicon was identified if it orits reverse and complemented sequence contained both forward and reverseprimer sequences. Occurrence of a unique sequence in a sample wasrecorded as its abundance. Percent indel or gene edit was calculated bydividing the number of reads with the edit or indel by the total numberof sequenced reads, and then multiplying by 100.

Sequence of CRISPR-Cas Photospacers (SEQ ID NOS 28, 25, and 44,Respectively, in Order of Appearance)

FIG. Name Sequence (5′ to 3′) Reference BC-1 CTCGTGACCACCTTCACCCA1a, 1b, 2a, 2c BC-2 GTCGTGCTGGTTCATGTGGT 2b BC-3 GGCTGAAGCACTGCACGGCG 2d

TALEN Binding Domain Sequences (SEQ ID NOS 45, 46, 38, and 39,Respectively, in Order of Appearance)

Paper FIG. Name Sequence (5′ to 3′) Reference BT-1Left arm: TGGTCGGGGTAGGGGCTGA 3a; 3b Right arm: TCGTGACCACCTTCACCCALuET-1 Left arm: TGGAACAGCTATGCGTCCG 3c; 3dRight arm: TGAGTTGCCTCCAGCGGCT

GRON Sequences used (SEQ ID NOS 37, 37, 24, 47, 26, 48, 19, 22, 36, 32,32, 49, 50, and 51, Respectively, in Order of Appearance)

FIG. Name Sequence (5′ to 3′) Chemistry Reference CG1GCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGG Unmodified 2a CG2G^C*T*GAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGA^G*G^G (^(x)) = 3PS 2a CG3C^(x)C*C^(x)TCGTGACCACCTTCACCTACGGCGTGCAGTGCTTC^(x)A*G*C (*) = 3PS 2d,3a CG4 VCCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCH V = CY3; H = 3′DMT dC2d CG5 G*T*G*ACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCC (*) = 3PS 2bAGACCACATGAAG*C^(x)A^(x)G CG6A^A^G*ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTT (^) = 3PS 1b. 2cGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCG^(x)C*C^(x)C CG7G^(x)G*G^(x)CGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA (*) = 3PS 3bTCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC AGGAGCGCACCAT*C*T*T CG8G^(x)G^G^(x)CGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCA (^) = 3PS 2bTCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCAGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC AGGAGCGCACCAT*C*T*T CG9A*A*G^(x)ATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTT (*) = 3PS 1aGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTACGTAAACGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGT GGCATCGCCCTCG*C*C^C CG10(a)agauggugcGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTG Lower Case = RNA 2cAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTG bases: (base) = 2′-O-Me;CACGCCGTAGGTGAAGGTGGTCACGAGGGTGGGCCAGGGCACGGGCA Upper Case = DNA basesGGCTTGCCGGTGGTGCAGATAACTTCAGGGTCAGCTTGCCGTAGGTG GCATCGCCCTCGCCC CG11(a)(a)(g)(a)(u)(g)(g)(u)(g)cGCTCCTGGACGTAGCCTTC Lower Case = RNA 2cGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGTGC bases: (base) = 2′-O-Me;AGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCC Upper Case = DNA basesCG12 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAA V = CY3; H = 3′DMTdC 3c, 3d TGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTG CPGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCAGCT TCTTH andVCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCAGCT TCTTH CG13G*C^(x)T*GAAGCACTGCACGCCGTGGGTGAAGGTGGTCACGA^(x)G*G^(x)G (*) = 3PSExtended Data FIG. 1

Statistical Snalysis

Statistical significance was determined using a Student's t-test withtwo-tailed distribution. P-values<0.05 were considered as significant.Data are shown as mean and SEM.

Results

CRISPR-Cas nuclease activity and gene editing in A. thalianaprotoplasts.

Engineered nucleases such as TAL effector nucleases (TALENs) andclustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease Cas9 (CRISPR-Cas9) can be programmed totarget and cleave double-stranded DNA with high specificity. TALENsconsist of two arms, both having a TAL effector-like DNA binding domain(TALE) linked to a catalytic DNA nuclease domain of FokI. The TALEdomains guide the TALEN arms to specific sites of DNA allowing fordimerization of the FokI endonucleases and subsequent generation of adouble strand break (DSB). The CRISPR-Cas9 system consists of a twocomponents; a Streptococcus pyogenes Cas9 nuclease (SpCas9) and achimeric fusion of two RNAs (crRNA and tracrRNA) referred to as anengineered single guide (sgRNA). The sgRNA supports targeted nucleicacid specificity for Cas9 through base pairing of its first twenty 5′bases with the DNA target, subsequently resulting in a site-specificDSB.

In plants, DSBs are typically repaired by the error prone non-homologousend joining (NHEJ) DNA repair pathway resulting in random deletionsand/or insertions (indels) at the site of repair. In contrast precisiongenome editing, relies on nuclease induced DSBs near the targeted changeto be repaired by homology directed repair (HDR), a repair pathway thatis more precise than NHEJ due to the requirement of a homologoustemplate DNA—in most cases sister chromatid. By harnessing the HDRpathway, it is possible to use an engineered oligonucleotide as therepair template to edit DNA specifically, when cleaved by nucleases ornon-specifically when used in combination with double strand breakinducing antibiotics.

FIG. 16 depicts CRISPR-Cas9 nuclease activity in Arabidopsis thalianaprotoplasts derived from the BFP transgenic model system in which astably integrated BFP gene can be converted to GFP by editing the codonencoding H66 (CAC→TAC H66Y). When cells were treated with CRISPR-Cas9(BC-1), NHEJ induced indels were produced at a frequency of 0.79% nearthe H66 locus of the BFP gene by deep sequencing (FIG. 16a ). Themajority of indels were single bp and none longer than 9 bp. Conversely,cells treated with GRON only or mock-treated did not exhibit indels(data not shown). These results show that CRISPR-Cas9 nuclease canactively target the BFP gene in this transgenic model system.

With regard to the effectiveness of CRISPR-Cas9 in combination with GRONto mediate BFP to GFP gene editing in protoplasts derived from ourtransgenic model system, a 7.4-fold improvement in BFP to GFP editingwas observed when both CRISPR-Cas9 and phosphorothioate (PS) modifiedGRONs (CG6) are introduced concurrently when compared to GRON alone orCRISPR-Cas9 alone treatments (FIG. 16b ). These results demonstrate thatintroducing CRISPR-Cas9 with PS modified GRONs into Arabidopsisprotoplasts significantly increase the frequency of BFP to GFP geneediting.

GRONs containing three adjacent PS modifications (herein refer to as3PS) at both the 5′ and 3′ ends positively effect BFP to GFP editingwhen compared to an unmodified GRON. The 3PS modified GRON (CG2), whencombined with CRISPR-Cas9 (BC-1), is more efficacious at BFP to GFPediting when compared to an unmodified GRON template (CG1; FIG. 17a ).In addition, a positive correlation between editing and GRON length(FIG. 17b ) was observed. Taken together, these results show that bothGRON modification and length can greatly improve the frequency of geneediting in plants such as Arabidopsis in the presence of CRISPR-Cas9.

When either the 201 nucleobase (nb) 3PS modified GRON (CG6), or the 201nb 2′-O-methyl modified GRONs (CG9) or (CG10), consisting of the firstten 5′ bases as RNA with either the first RNA base or the first 9 RNAbases modified with 2′-O-methyl are introduced along with CRISPR-Cas9(BC-1) into Arabidopsis protoplasts, no statistical difference in BFP toGFP editing between them was observed (FIG. 17c ). Similarly, wheneither the 201 nb 3PS modified GRON (CG3) or the 201 nb Cy3 modifiedGRON (CG4), comprising of a 5′ Cy3 and an 3′ idC reverse base, wereintroduced along with CRISPR-Cas9 (BC-3) into Arabidopsis protoplasts,no statistical difference in editing frequencies was observed (FIG. 17d). Overall, these data show that diverse GRON modifications can greatlyimprove the frequency of gene editing in Arabidopsis in the presence ofCRISPR-Cas9.

Based on these results with CRISPR-Cas9 and modified GRONs, it wasdetermined if modified GRONs coupled with TALEN pairs targeting the BFPgene result in improved BFP to GFP gene editing as well. To first showeffective nuclease activity at the BFP locus Arabidopsis protoplastswere treated with TALENs (BT-1) and found 0.51% indels at or near theexpected cleavage site by deep sequencing—indicating that TALENs are asactive as CRISPR-Cas9 in this model system (FIG. 18a ). The majority ofdeletions were >10 bp but less than 50 bp, while insertions,significantly less abundant than deletions were three bp or less. Nextwe examined the effectiveness of TALENs coupled with modified GRON toedit BFP to GFP. A 9.2-fold improvement in the frequency of BFP to GFPediting was observed when both the BFP TALEN (BT-1) and 3PS GRON (CG7)are introduced when compared to 3PS GRON alone (FIG. 18b ). Similar tothe CRISPR-Cas experiments described above, these results demonstratethat introducing TALENs with 3PS modified GRONs into Arabidopsisprotoplasts also significantly increases the frequency of BFP to GFPgene editing.

The EPSPS (5′-enolpyruvylshikimate-3-phosphate synthase) loci in Linumusitatissimum (Common flax) was also used as a target in this system.The EPSPS genes encode an enzyme in the shikimate pathway thatparticipates in the biosynthesis of the aromatic amino acidsphenylalanine, tyrosine and tryptophan. In plants, EPSPS is a target forthe herbicide, glyphosate, where it acts as a competitive inhibitor ofthe binding site for phosphoenolpyruvate.

TALENs targeting a site near two loci (T97I and P101A) in L.usitatissimum that when edited will render EPSPS tolerant to glyphosate(Extended Data FIG. 18b ) were selected. Delivering TALEN (LuET-1)together with a 144 nb 5′ Cy3 modified GRON (CG11) containing thetargeted changes at T97I and P101A into protoplasts, gene editingfrequencies of 0.19% at both loci and indel frequency at 0.5% seven daysafter introduction were observed (FIG. 18c, 18d ). The majority ofindels were 10 bp or less (FIG. 18c ). These results demonstrate thatintroducing TALENs with Cy3 modified GRONs into L. usitatissimumprotoplasts significantly increase the frequency of EPSPS gene editingand further that multiple nucleotide edits can be realized with a singleGRON.

Example 20: Effect of Two Members of the Bleomycin Family of Antibioticson Conversion

The purpose of this series of examples was to evaluate the effect ofantibiotics on conversion efficiencies.

Methods

Protoplasts from an Arabidopsis thaliana line with multiple copies of ablue fluorescent protein gene were treated with GRON as described inExample 1, with the following modification: before the addition of GRON,the protoplasts were kept for 90 minutes on ice in a solution of TM(14.8 mM MgCl₂×6H₂O, 5 mM 2-(N-morpholino)ethanesulfonic acid, 572 mMmannitol), supplemented with 0, 250, or 1000 μg/ml Zeocin™ orphleomycin. The pH of the solutions was adjusted to 7.0. The percentageof green-fluorescing cells resulting from BFP to GFP conversion wasevaluated by flow cytometry as described in Example 1.

Results

Zeocin and phleomycin at both concentrations used (250 and 1000 μg/ml)resulted in an increase in BFP to GFP gene editing (see FIG. 19).Green-fluorescing cells resulting from BFP to GFP gene editing wereobserved five days after GRON delivery (FIG. 20).

REFERENCES

1. LeCong et al 2013 Science : vol. 339 no. 6121 pp. 819-823.

2. Jinek et al 2012 Science. 337:816-21

3. Wang et al 2008 RNA 14: 903-913

4. Zhang et al 2013. Plant. Physiol. 161: 20-27

Example 21: CRiSPRs and GRONs in Rice

The purpose of this experiment is to demonstrate ACCase conversion inOryza sativa at 120 hours after PEG delivery of CRISPR-Cas plasmids andGRONs into protoplasts. The CRISPR-Cas used in this experiment targetsthe accase gene in the rice genome by introducing into protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target genes, where Cas9 creates adouble-stranded break in the accase gene and the GRON is used as atemplate to convert the accase gene in a site-directed manner.

Methods

Rice protoplasts were isolated from calli. The CRISPR-Cas encodedplasmids contains the corn ubiquitin promoter driving the Cas9 codingsequence with an rbcSE9 terminator and a rice U6 promoter promoterdriving the sgRNA with a poly-Tio terminator (“T10” disclosed as SEQ IDNO: 21). The CRISPR-Cas plasmids were introduced into protoplasts by PEGmediated delivery at a final concentration of 0.05 μg/μl. GRONs with thefollowing sequence, 5′ V C TGA CCT GAA CTT GAT CTC AAT TAA CCC TTG CGGTTC CAG AAC ATT GCC TTT TGC AGT CCT CTC AGC ATA GCA CTC AAT GCG GTC TGGGTT TAT CTT GCT TCC AAC GAC AAC CCA AGC CCC TCC TCG TAG CTC TGC AGC CATGGG AAT GTA GAC AAA GGC AGG CTG ATT GTA TGT CCT AAG GTT CTC AAC AAT AGTCGA GCC H 3′ (SEQ ID NO:52), were used at a final concentration of 0.8Protoplasts were embedded in agarose (2.5×106 cells/ml), cultured inliquid medium, and incubated in a rotatory shaker (60 rpm) in the darkat 28° C. Individual samples were analyzed by NGS, 120 hours afterCRISPR-Cas plasmid and/or GRON delivery, to determine the percentage ofcells (DNA reads) carrying the ACCase conversion and having indels inthe accase gene.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from the same plasmid. The sgRNA is a fusion of CRISPR RNA(crRNA) and trans-activating crRNA (tracrRNA). The crRNA region containsthe spacer sequence (5′-ACGAGGAGGGGCTTGGGTTGTGG-3 (SEQ ID NO:53)) usedto guide the Cas9 nuclease to the target gene. In this experiment theCRISPR targets the accase gene.

Results

At 120 h, rice protoplasts have 0.026% ACCase conversion as determinedby Next Generation Sequencing. GRON only controls with no CRISPR-Casshowed minimal Accase conversion of 0.002% at 120 hours and theuntreated controls showed no conversion. Additionally, these data showthat the CRISPR-Cas is active and able to cleave the ACCase target geneand form indels of 8.0%.

REFERENCES

5. LeCong et al 2013 Science : vol. 339 no. 6121 pp. 819-823.

6. Jinek et al 2012 Science. 337:816-21

7. Wang et al 2008 RNA 14: 903-913

8. Zhang et al 2013. Plant 161: 20-27

Example 22: CRISPRs and GRONs in Rice

Summary: Targeted ACCase mutations have been identified in nineteenweek-old calli by PCR and DNA sequencing analyses.

Introduction

The purpose of this experiment is to demonstrate ACCase conversion inOryza sativa calli after PEG delivery of CRISPR-Cas plasmids and GRONsinto protoplasts. The CRISPR-Cas used in this experiment targets theACCase gene in the rice genome by introducing into protoplastsplasmid(s) encoding the Cas9 gene and a sgRNAs. The sgRNA is a fusion ofCRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the target gene, where Cas9 creates a either adouble-stranded break or a nick at a targeted location in the ACCasegene and the GRONs are used as a template to convert the ACCase gene ina site-directed manner.

Results

Targeted OsACCase mutations described in the tables below, have beenidentified in nineteen week-old calli by PCR and DNA sequencinganalyses.

Methods

Rice protoplasts were isolated from suspension cultures initiated frommature seed-derived embryogenic calli. The CRISPR-Cas plasmids withGRONs at a final concentration of 0.05 μg/μl and 0.8 respectively, wereintroduced into protoplasts by PEG mediated delivery method. Anexemplary range for CRISPR-Cas plasmids at a final concentrationinclude, but is not limited to 0.01 to 0.2 μg/μl. An exemplary range forGRON at a final concentration include, but is not limited to 0.01 to 4The CRISPR-Cas encoded plasmids contains the corn ubiquitin promoterdriving the Cas9 coding sequence with an rbcSE9 terminator and a rice U6promoter promoter driving the sgRNA with a poly-Tio terminator (“T10”disclosed as SEQ ID NO: 21). Sequence information of the GRONs aredescribed in Table 3. Following the PEG treatment, protoplasts wereembedded in agarose (1.25×10⁶ cells/ml), cultured in liquid medium, andincubated in a rotatory shaker (60 rpm) in the dark at 28° C. Anexemplary range for embedding protoplasts in agarose include, but is notlimited to 0.625×10⁶ to 2.5×10⁶ cells/ml. Samples from each treatmentwere analyzed by Next Generation Sequencing after 4 weeks postCRISPR-Cas plasmid and/or GRON treatment to determine the proportion ofcells (DNA reads) carrying the ACCase conversion. Microcalli fromconverted treatments were released onto solid selection mediumcontaining clethodim (0.25-0.5 μM) or sethoxydim (2.5 μM). Individualcallus lines growing on this selection medium after 19 weeks of culturewere analyzed by in-house screening methods as well as DNA sequencing inorder to identify individual calli containing the targeted ACCaseconversions.

The CRISPR consists of two components: the Cas9, such as a plantcodon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNA both ofwhich were expressed from plasmid(s). Cas9 and sgRNA can also bedelivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusionof CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAregion contains the spacer with sequences described in Table 2, whichwere used to guide the Cas9 nuclease to the target gene. In thisexperiment the CRISPR targets the rice ACCase gene.

List of conversions in OsACCase at two different locations within thegene, Site 1 and

Site 2. For each site, all combinations of conversion events arepossible.

OsACCase Site1 Site 2 I1781A D2078G I1781L D2078K I1781M D2078T I1781NS2079F I1781S K2080E I1781T C2088F I1781V C2088G G1783C C2088H A1786PC2088K C2088L C2088N C2088P C2088Q C2088R C2088S C2088T C2088V C2088W

List of CRISPR-Cas gRNA spacer sequences used in this experiment. Spacerlength may vary up to ±20 bp. Mismatched within the spacer sequence canbe tolerated up to 10 bp.

SEQ Sample Spacer RNA Sequence OsACCase ID ID (5' to 3') Site 1 Site 2NO: 1 CAUAAGAUGCAGCUAGACAG X 54 2 AGCUAGACAGUGGUGAAAUU X 55 3UAGACAGUGGUGAAAUUAGG X 56 4 AGACAGUGGUGAAAUUAGGU X 57 5GUGGGUUAUUGAUUCUGUUG X 58 6 UGGGUUAUUGAUUCUGUUGU X 59 7UAUUGAUUCUGUUGUGGGCA X 60 8 UCUGUUGUGGGCAAGGAAGA X 61 9GUGGGCAAGGAAGAUGGACU X 62 10 CAAGGAAGAUGGACUUGGUG X 63 11CUUGGUGUGGAGAAUAUACA X 64 12 CUAUUGCCAGUGCUUAUUCU X 65 13UGCUUAUUCUAGGGCAUAUA X 66 14 UUUACACUUACAUUUGUGAC X 67 15UUUGUGACUGGAAGAACUGU X 68 16 ACUGGAAGAACUGUUGGAAU X 69 17GGAGCUUAUCUUGCUCGACU X 70 18 AUAUGCCCUAGAAUAAGCAC X 71 19GAUAAGAUGCAGCUAGACAG X 72 20 GGCUAGACAGUGGUGAAAUU X 73 21GAGACAGUGGUGAAAUUAGG X 74 22 GGACAGUGGUGAAAUUAGGU X 75 24GGGGUUAUUGAUUCUGUUGU X 76 25 GAUUGAUUCUGUUGUGGGCA X 77 26GCUGUUGUGGGCAAGGAAGA X 78 28 GAAGGAAGAUGGACUUGGUG X 79 29GUUGGUGUGGAGAAUAUACA X 80 30 GUAUUGCCAGUGCUUAUUCU X 81 31GGCUUAUUCUAGGGCAUAUA X 82 32 GUUACACUUACAUUUGUGAC X 83 33GUUGUGACUGGAAGAACUGU X 84 34 GCUGGAAGAACUGUUGGAAU X 85 36GUAUGCCCUAGAAUAAGCAC X 86 37 CGACUAUUGUUGAGAACCUU X 87 38UGCCUUUGUCUACAUUCCCA X 88 39 CCCAUGGCUGCAGAGCUACG X 89 40AUGGCUGCAGAGCUACGAGG X 90 41 UGGCUGCAGAGCUACGAGGA X 91 42GGCUGCAGAGCUACGAGGAG X 92 43 CAGAGCUACGAGGAGGGGCU X 93 44AGAGCUACGAGGAGGGGCUU X 94 45 ACGAGGAGGGGCUUGGGUUG X 95 46GCAUUGAGUGCUAUGCUGAG X 96 47 UAUGCUGAGAGGACUGCAAA X 97 48GACUGCAAAAGGCAAUGUUC X 98 49 GGCAAUGUUCUGGAACCGCA X 99 50GCAAUGUUCUGGAACCGCAA X 100 51 GGUUAAUUGAGAUCAAGUUC X 101 52UGAGAUCAAGUUCAGGUCAG X 102 53 GUUCAGGUCAGAGGAACUCC X 103 54AACUCCAGGAUUGCAUGAGU X 104 55 CAAGCCGACUCAUGCAAUCC X 105 56UUGAUCUCAAUUAACCCUUG X 106 57 UCUCAGCAUAGCACUCAAUG X 107 58GCAUAGCACUCAAUGCGGUC X 108 59 CAUAGCACUCAAUGCGGUCU X 109 60UCCUCGUAGCUCUGCAGCCA X 110 61 AGCCAUGGGAAUGUAGACAA X 111 62AUGGGAAUGUAGACAAAGGC X 112 63 CAGGCUGAUUGUAUGUCCUA X 113 64GGACUAUUGUUGAGAACCUU X 114 65 GGCCUUUGUCUACAUUCCCA X 115 66GCCAUGGCUGCAGAGCUACG X 116 67 GUGGCUGCAGAGCUACGAGG X 117 68GGGCUGCAGAGCUACGAGGA X 118 69 GAGAGCUACGAGGAGGGGCU X 119 70GGAGCUACGAGGAGGGGCUU X 120 71 GCGAGGAGGGGCUUGGGUUG X 121 72GAUGCUGAGAGGACUGCAAA X 122 73 GGAGAUCAAGUUCAGGUCAG X 123 74GACUCCAGGAUUGCAUGAGU X 124 75 GAAGCCGACUCAUGCAAUCC X 125 76GUGAUCUCAAUUAACCCUUG X 126 77 GCUCAGCAUAGCACUCAAUG X 127 78GAUAGCACUCAAUGCGGUCU X 128 79 GCCUCGUAGCUCUGCAGCCA X 129 80GGCCAUGGGAAUGUAGACAA X 130 81 GUGGGAAUGUAGACAAAGGC X 131 82GAGGCUGAUUGUAUGUCCUA X 132

A list of GRON sequences suitable for use in this experiment areprovided in the table below (V=CY3; H=3′DMT dC CPG).

1 VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATGCTCATGGAAGTGCTGCTATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH (SEQ ID NO: 133) 2VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATAGCAGCACTTCCATGCAGATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH (SEQ ID NO: 134) 3VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATATACATTGCAGTGCTGCTATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH (SEQ ID NO: 135) 4VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATAGCAGCACTGCAATGTATATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH (SEQ ID NO: 136) 5VGTCATAGCACATAAGATGCAGCTAGACAGTGGTGAAATTAGGTGGGTTATTGATTCTGTTGTGGGCAAGGAAGATGGACTTGGTGTGGAGAATATACATGGAAGTGCTCCAATTGCCAGTGCTTATTCTAGGGCATATAAGGAGACATTTACACTTACATTTGTGACTGGAAGAACTGTTGGAATAGGAGCTTATCTTGCTCH (SEQ ID NO: 137) 6VGAGCAAGATAAGCTCCTATTCCAACAGTTCTTCCAGTCACAAATGTAAGTGTAAATGTCTCCTTATATGCCCTAGAATAAGCACTGGCAATGGTAGCACTTCCATGTATATTCTCCACACCAAGTCCATCTTCCTTGCCCACAACAGAATCAATAACCCACCTAATTTCACCACTGTCTAGCTGCATCTTATGTGCTATGACH (SEQ ID NO: 138) 7VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATCTTGCTTCCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH (SEQ ID NO: 52) 8VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGGTAGCAAGATAAACCCAGACCGCATTGAGTGCTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH (SEQ ID NO: 139) 9VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATCTTAAAATCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH (SEQ ID NO: 140) 10VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGCGCTTGGGTTGTGGTTGATAGCAAGATAAACCCAGACCGCATTGAGAGGTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH (SEQ ID NO: 141) 11VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGATAGCGAAATAAACCCAGACCGCATTGAGTGCTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH (SEQ ID NO: 142) 12VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATAGCACTCAATGCGGTCTGGGTTTATTTCGCTATCAACCACAACCCAAGCGCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH (SEQ ID NO: 143) 13VGGCTCGACTATTGTTGAGAACCTTAGGACATACAATCAGCCTGCCTTTGTCTACATTCCCATGGCTGCAGAGCTACGAGGAGGGGCTTGGGTTGTGGTTGATAGCAAGATAAACCCAGACCGCATTGAGCGTTATGCTGAGAGGACTGCAAAAGGCAATGTTCTGGAACCGCAAGGGTTAATTGAGATCAAGTTCAGGTCAGH (SEQ ID NO: 144) 14VCTGACCTGAACTTGATCTCAATTAACCCTTGCGGTTCCAGAACATTGCCTTTTGCAGTCCTCTCAGCATATTGCTCAATGCGGTCTGGGTTTATCTTGCTATCAACGACAACCCAAGCCCCTCCTCGTAGCTCTGCAGCCATGGGAATGTAGACAAAGGCAGGCTGATTGTATGTCCTAAGGTTCTCAACAATAGTCGAGCCH (SEQ ID NO: 145)

Example 23: CRISPRs and GRONs in Flax

Summary: Targeted LuEPSPS mutations have been identified in fourweek-old calli by PCR and DNA sequencing analyses.

Introduction

The purpose of this experiment is to demonstrate conversion of the EPSPSgenes in the Linum usitatissimum genome in shoot tip derived protoplastsby PEG mediated delivery of CRISPR-Cas plasmids and GRONs. TheCRISPR-Cas and GRONs used in this experiment target the EPSPS genes inthe flax genome. The CRISPR consists of two components: a Cas9, such asa plant codon-optimized Streptococcus pyogenes Cas9 (SpCas9) and sgRNAboth of which are expressed from plasmid(s). Cas9 and sgRNA can also bedelivered by mRNA/RNA or protein/RNA respectively. The sgRNA is a fusionof CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNAguides the Cas9 to the targeted genes, where Cas9 creates a either adouble-stranded break or nick in the EPSPS genes and the GRONs are usedas a template to convert the EPSPS genes in a site-directed manner.

Results

Targeted LuEPSPS (T97I and/or the P101A, P101T or P101S and/or the G96A)mutations have been identified in four week-old calli by PCR and DNAsequencing analyses. Shoots have been regenerated from these convertedcalli.

Methods

Flax protoplasts were isolated from shoot tips obtained from in vitrogerminated seedlings. The CRISPR-Cas encoded plasmids contain the MASpromoter driving the Cas9 coding sequence with an rbcSE9 terminator andthe Arabidopsis thaliana U6 promoter driving the sgRNA with a poly-Tioterminator (“T10” disclosed as SEQ ID NO: 21). The CRISPR-Cas plasmidswere introduced into protoplasts by PEG mediated delivery at a finalconcentration of 0.05 μg/μl. GRONs targeting each of the two flaxLuEPSPS genes (Table 2) were used at a final concentration of 4.0 μM. Anexemplary range for CRISPR-Cas plasmids at a final concentrationinclude, but is not limited to 0.01 to 0.2 μg/μl. An exemplary range forGRON at a final concentration include, but is not limited to 0.01 to 4μM. Protoplasts were embedded in alginate beads (5×10⁵ cells/ml),cultured in liquid medium, and incubated in a rotatory shaker (30 rpm)in the dark at 25° C. An exemplary range for embedding protoplasts inalginate beads include, but is not limited to 3.0×10⁵ to 7.5×10⁵cells/ml. Microcalli developed from individual cells were analyzed byNext Generation Sequencing, 3 and 7 weeks after CRISPR-Cas plasmid andGRON delivery, to determine the proportion of cells (DNA reads) carryingthe targeted mutations in the LuEPSPS genes. Larger calli were grownfrom 8-week-old converted microcalli plated over solid regenerationmedium, and shoots started differentiating from regenerated calli afterabout 4-8 weeks. Converted calli and shoots with the targeted EPSPS genemutations were identified by PCR and DNA sequencing analyses.

The CRISPR consists of two components: the plant codon-optimizedStreptococcus pyogenes Cas9 (SpCas9) and sgRNA both of which wereexpressed from plasmid(s). The sgRNA is a fusion of CRISPR RNA (crRNA)and trans-activating crRNA (tracrRNA). The crRNA region contains thespacer with sequences described in the table below, which were used toguide the Cas9 nuclease to the EPSPS targeted genes.

List of CRISPR-Cas gRNA spacer sequences used in this experiment. Spacerlength may vary up to ±20 bp. Mismatched within the spacer sequence canbe tolerated up to 10 bp.

LuEPSPS SEQ Sample Spacer RNA Sequence Genes ID ID (5′ to 3′) 1 2 NO: 1CAGAAGCGCGCCAUUGUUGA X X 146 2 CGCGCCAUUGUUGAAGGUUG X 147 3CGCGCCAUUGUUGAAGGUCG X 148 4 GCCAUUGUUGAAGGUUGUGG X 149 5GCCAUUGUUGAAGGUCGUGG X 150 6 AGGUUGUGGUGGUGUGUUUC X 151 7AGGUCGUGGUGGUGUGUUUC X 152 8 UGUGGUGGUGUGUUUCCGGU X 153 9CGUGGUGGUGUGUUUCCGGU X 154 10 UGUGUUUCCGGUCGGUAAAC X X 155 11UGUUUCCGGUCGGUAAACUG X 156 12 AACGAUAUUGAACUUUUCCU X 157 13AACGAUAUCGAACUUUUCCU X 158 14 GAACUUUUCCUUGGAAAUGC X X 159 15ACAGCUGCUGUAACAGCCGC X X 160 16 GCUGCUGUAACAGCCGCUGG X X 161 17AACUCAAGCUACAUACUCGA X 162 18 AACUCAAGCUACAUACUCGA X 163 19CGAAUGAGAGAGAGACCAAU X 164 20 CGAAUGAGAGAGAGACCGAU X 165 21AGAGAGACCAAUUGGAGAUU X 166 22 CCAAUUGGAGAUUUGGUUGU X 167 23CCGAUUGGAGAUUUAGUUGU X 168 24 CCAACAACCAAAUCUCCAAU X 169 25CCAACAACUAAAUCUCCAAU X 170 26 AUUGGUCUCUCUCUCAUUCG X 171 27AUCGGUCUCUCUCUCAUUCG X 172 28 GUAGCUUGAGUUGCCUCCAG X X 173 29GCUGUUACAGCAGCUGUCAG X X 174 30 UAGCUGUUCCAGCAUUUCCA X X 175 31UUCUUCGCCAGUUUACCGAC X 176 32 UUCUUCCCCAGUUUACCGAC X 177 33ACCACCACAACCUUCAACAA X 178 34 ACCACCACGACCUUCAACAA X 179 35GAGAAGCGCGCCAUUGUUGA X X 180 36 GGCGCCAUUGUUGAAGGUUG X 181 37GGCGCCAUUGUUGAAGGUCG X 182 38 GGGUUGUGGUGGUGUGUUUC X 183 39GGGUCGUGGUGGUGUGUUUC X 184 40 GGUGGUGGUGUGUUUCCGGU X 185 41GGUGGUGGUGUGUUUCCGGU X 186 42 GGUGUUUCCGGUCGGUAAAC X X 187 43GGUUUCCGGUCGGUAAACUG X 188 44 GACGAUAUUGAACUUUUCCU X 189 45GACGAUAUCGAACUUUUCCU X 190 46 GCAGCUGCUGUAACAGCCGC X X 191 47GACUCAAGCUACAUACUCGA X 192 48 GACUCAAGCUACAUACUCGA X 193 49GGAAUGAGAGAGAGACCAAU X 194 50 GGAAUGAGAGAGAGACCGAU X 195 51GGAGAGACCAAUUGGAGAUU X 196 52 GCAAUUGGAGAUUUGGUUGU X 197 53GCGAUUGGAGAUUUAGUUGU X 198 54 GCAACAACCAAAUCUCCAAU X 199 55GCAACAACUAAAUCUCCAAU X 200 56 GUUGGUCUCUCUCUCAUUCG X 201 57GUCGGUCUCUCUCUCAUUCG X 202 58 GAGCUGUUCCAGCAUUUCCA X X 203 59GUCUUCGCCAGUUUACCGAC X 204 60 GUCUUCCCCAGUUUACCGAC X 205 61GCCACCACAACCUUCAACAA X 206 62 GCCACCACGACCUUCAACAA X 207

A list of GRON sequences suitable for use in this experiment areprovided in the table below (V=CY3; H=3′DMT dC CPG).

1 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGCTATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 208)2 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTGTGGCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGCCAGTTTA CCGACCGH (SEQ ID NO: 209)3 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCCTGGAGGCAACTCAAGGTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 49)4 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGCCAGTTTA CCGACCGH (SEQ ID NO: 210)5 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTTCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 211)6 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGAACGCATAGCAATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGCCAGTTTA CCGACCGH (SEQ ID NO: 212)7 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTACTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 213)8 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGTACGCATAGCAATTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGCCAGTTTA CCGACCGH (SEQ ID NO: 214)9 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 215)10 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 216)11 VCGGTCGGTAAACTGGCGAAGAACGATATTGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTCCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 217)12 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGGACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTGTTCCAGCATTTCCAAGGAAAAGTTCAATATCGTTCTTCGCCAGTTTA CCGACCGH (SEQ ID NO: 218)13 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGCTATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 219)14 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTGTTGCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 220)15 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 221)16 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGCACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 222)17 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATTGCTATGCGTTCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 223)18 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGAACGCATAGCGATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 224)19 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTACTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 225)20 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGTGTACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 226)21 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATCGCTATGCGTGCGCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 227)22 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGCGGACGCATAGCTATTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 228)23 VCGGTCGGTAAACTGGGGAAGAACGATATCGAACTTTTCCTTGGAAATGCTGGAATAGCTATGCGTGCTCTGACAGCTGCTGTAACAGCCGCTGGAGGCAACTCAAGGTTCCTTCCCTCAACTCCTTCCAGCCTTTCA GCTTCTTH (SEQ ID NO: 229)24 VAAGAAGCTGAAAGGCTGGAAGGAGTTGAGGGAAGGAACCTTGAGTTGCCTCCAGCGGCTGTTACAGCAGCTGTCAGGGAACGCATAGCTGTTCCAGCATTTCCAAGGAAAAGTTCGATATCGTTCTTCCCCAGTTTA CCGACCGH (SEQ ID NO: 230)

One skilled in the art readily appreciates that the present disclosureis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the disclosure disclosedherein without departing from the scope and spirit of the disclosure.

The disclosure illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure claimed. Thus, it should be understood thatalthough the present disclosure has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement, and variation of the disclosuresdisclosed may be resorted to by those skilled in the art, and that suchmodifications, improvements and variations are considered to be withinthe scope of this disclosure. The materials, methods, and examplesprovided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of thedisclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

1. A method of causing one or more targeted genetic changes in anendogenous target gene in the genome of a plant cell, said methodcomprising: delivery to the plant cell of a DNA cutter which inducessingle or double strand breaks and a gene repair oligonucleobase (GRON)configured to mediate introduction of the one or more targeted geneticchanges within the endogenous target gene in the genome of the plantcell, wherein the GRON comprises one or more 5′ blocking substituents,wherein the 5′ blocking substituents comprise a terminal2′-O-methoxyethyl nucleotide, 2′-O-methyl nucleotide or Cy3 group,wherein the plant cell is non-transgenic with respect to the targetedgenetic change, and wherein the DNA cutter is selected from the groupconsisting of a CRISPR, a TALEN, a zinc finger, meganuclease, and aDNA-cutting antibiotic.
 2. The method of claim 1, wherein said DNAcutter is a CRISPR.
 3. The method of claim 1, wherein said DNA cutter isa TALEN.
 4. The method of claim 1, wherein said DNA cutter is one ormore DNA-cutting antibiotics selected from the group consisting ofbleomycin, zeocin, phleomycin, tallysomycin and pepleomycin.
 5. Themethod of claim 4, wherein said DNA cutter is zeocin.
 6. The method ofclaim 1, wherein said GRON is single stranded.
 7. The method of claim 1,wherein the GRON comprises a 3′ reverse base blocking group.
 11. Themethod of claim 1, wherein said GRON has a wobble base pair relative tothe target sequence for the genetic change.
 12. The method of claim 1,wherein said GRON is between 20 and 1000 nucleotides in length.